Dose of an angiogenic factor and method of administering to improve myocardial blood flow

ABSTRACT

The present invention has multiple aspects. In one aspect, the present invention is directed to a unit dose pharmaceutical composition comprising from about 5 ng/dose to less than 135,000 ng of an angiogenic agent, typically from 5 ng to 67,500 ng. Preferably, the angiogenic agent is FGF, more preferably it is basic FGF (FGF-2). In its second aspect, the present invention is directed to a method for inducing angiogenesis, or increasing myocardial perfusion or vascular density in a patient&#39;s heart, comprising administering directly into the myocardium in an area in need, as a single injection or a series of injections, a unit dose of an angiogenic agent. It is also within the scope of the present invention that a plurality of unit dose compositions be administered directly into the myocardium at a plurality of sites in need of angiogenesis. In another aspect, the present invention is directed to a method for treating a patient for coronary artery disease, comprising administering directly into the myocardium in an area of need of angiogenesis in said patient, a unit dose (i.e., from about 5 ng to less than 135,000 ng) of an angiogenic agent. In yet another aspect, the present invention is directed to a method for treating a patient for a myocardial infarction, comprising administering directly into the myocardium in an area in need of angiogenesis in said patient, a unit dose (i.e., from about 5 ng to less than 135,000 ng) of an angiogenic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/395,541, filed Mar. 24, 2003, which is a continuation of U.S.application Ser. No. 09/637,471, filed Aug. 11, 2000, now abandoned,which claims the benefit of U.S. Provisional Application No. 60/148,746,filed Aug. 13, 1999, the contents of which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention is directed to a dose, including an ultra-lowdose, of an angiogenic factor, such as a fibroblast growth factor (FGF),platelet derived growth factor or a vascular endothelial growth factor,or an angiogenically active fragment or mutein thereof, and to a mode ofadministering the dose to obtain improved myocardial blood flow. Thepresent invention is also directed to a pharmaceutical compositioncomprising the dose of angiogenic factor and to a method foradministering the pharmaceutical composition to a heart, preferably ahuman heart, to improve myocardial function, blood flow, perfusionand/or vascular density. The present invention is useful because thedisclosed dose, pharmaceutical composition and method for itsadministration provide an alternative or adjunct to surgicalintervention for the treatment of coronary artery disease (CAD) and/orfurther provide a method for reducing post myocardial infarct (MI)injury in humans. Finally, the present invention includes a method fordetermining whether the administered angiogenic agent is having atherapeutic effect on the target tissues by assaying for a surrogatemarker.

BACKGROUND OF THE INVENTION

Coronary artery disease (atherosclerosis) is a progressive disease inhumans wherein one or more coronary arteries gradually become occludedthrough the buildup of plaque. The coronary arteries of patients havingthis disease are often treated by balloon angioplasty or the insertionof stents to prop open the partially occluded arteries. Ultimately,these patients are required to undergo coronary artery bypass surgery atgreat expense and risk. It would be desirable to provide such patientswith a treatment that would enhance coronary blood flow so as topreclude the need to undergo bypass surgery or angioplasty.

An even more critical situation arises in humans when a patient suffersa myocardial infarction, wherein one or more coronary arteries orarterioles becomes completely occluded, such as by a clot. There is animmediate need to regain circulation to the portion of the myocardiumserved by the occluded artery or arteriole. If the lost coronarycirculation is restored within hours of the onset of the infarction,much of the damage to the myocardium that is downstream from theocclusion can be prevented. The clot-dissolving drugs, such as tissueplasminogen activator (tPA), streptokinase, and urokinase, have beenproven to be useful in this instance. However, as an adjunct to the clotdissolving drugs, it would also be desirable to also obtain collateralcirculation to the damaged or occluded myocardium by angiogenesis.

Accordingly, it is an object of the present invention to provide a doseof an angiogenic agent and a mode of its administration to a human heartin need of angiogenesis that provides the human heart with cardiacangiogenesis while minimizing the risk of inducing angiogenesiselsewhere in the body, particularly in an undetected tumor. Moreparticularly, it is a further object of the present invention to providea therapeutic dose of an angiogenic factor and a mode of itsadministration to a human patient that provides the desired property ofcardiac angiogenesis, such as during the treatment of coronary arterydisease and/or post acute myocardial infarction, while minimizing thepossibility of an adverse angiogenic effect occurring elsewhere in thebody.

Angiogenic agents include the platelet derived growth factors (PDGF),vascular endothelial growth factor-A (VEGF-A), transforming growthfactor-β1 (TGF-β1) and the fibroblast growth factors. The fibroblastgrowth factors (FGF) are a family of at least eighteen structurallyrelated polypeptides (named FGF-1 to FGF-18) that are characterized by ahigh degree of affinity for proteoglycans, such as heparin. The variousFGF molecules range in size from 15-23 kD, and exhibit a broad range ofbiological activities in normal and malignant conditions including nervecell adhesion and differentiation [Schubert et al., J. Cell Biol.104:635-643 (1987)]; wound healing [U.S. Pat. No. 5,439,818 (Fiddes)];as mitogens toward many mesodermal and ectodermal cell types, as trophicfactors, as differentiation inducing or inhibiting factors [Clements, etal., Oncogene 8:1311-1316 (1993)]; and as an angiogenic factor [Harada,J. Clin. Invest., 94:623-630 (1994)]. Thus, the FGF family is a familyof pluripotent growth factors that stimulate to varying extentsfibroblasts, smooth muscle cells, epithelial cells and neuronal cells.

When any angiogenic agent (or factor) is released by normal tissues,such as in fetal development or wound healing, it is subject to temporaland spatial controls. However, many angiogenic agents are alsooncogenes. Thus, in the absence of temporal and spatial controls, theyhave the potential to stimulate tumor growth by providing angiogenesis.Accordingly, before any angiogenic agent is used as a medicament inhuman patients, consideration must be given to minimizing its angiogeniceffect on undetected tumors. As a result, it is an object of the presentinvention to provide a dosage of angiogenic agent and a mode of itsadministration that would provide localized angiogenesis in a targetedorgan but which would minimize the risk of enhancing angiogenesis in anundetected tumor elsewhere in the body.

Many of the angiogenic agents (e.g., PDGF, VEGF-A or FGF) have beenisolated and administered to various animal models of myocardialischemia with varying and often times opposite results. According toBattler et al., “the canine model of myocardial ischemia has beencriticized because of the abundance of naturally occurring collateralcirculation, as opposed to the porcine model, which ‘excels’ in itsrelative paucity of natural collateral circulation and its resemblanceto the human coronary circulation.” Battler et al., “IntracoronaryInjection of Basic Fibroblast Growth Factor Enhances Angiogenesis inInfarcted Swine Myocardium,” JACC, 22(7): 2001-6 (December 1993) at page2002, col. 1. Thus, those of ordinary skill in the art considered theporcine heart to be the model that excelled most in its resemblance tothe human heart. Further, Battler points out that “the dosage and modeof administration of bFGF [i.e., bovine FGF-2] may have profoundimplications for the biologic effect achieved.” Battler, et al., at page2005, col. 1. Thus, it is a further object of this invention to providea dosage and a mode of administration of an angiogenic agent that wouldprovide for the safe and efficacious treatment of CAD and/or post MIinjury in a human patient. More generally, it is an object of thepresent invention to provide a pharmaceutical composition and method foradministration that would induce angiogenesis in a human heart whileminimizing the risk of angiogenesis elsewhere in the body.

The various studies to date on angiogenic agents have administereddosages of the angiogenic agent in the range of 10 μg to 1500 μg. Forexample, Yanagisawa-Miwa, et al., “Salvage of Infarcted Myocardium byAngiogenesic Action of Basic Fibroblast Growth Factor,” Science,257:1401-1403 (1992), disclose infusing two 10 μg doses of humanrecombinant basic FGF (hrFGF-2) in 10 ml of saline over a one minuteperiod into the left circumflex coronary artery (LCX) of dogs afterinducing a myocardial infarction by inserting a thrombus into theadjacent left ascending coronary artery (LAD). Yanagisawa-Miwa furtherdiscloses that as a result of the intracoronary administration of atotal of 20 μg of hrFGF-2 in this canine model, “vessel formationoccurred within 1 week after administration of bFGF.” Yanagisawa-Miwa atpage 1403. Banai et al., “Angiogenic-Induced Enhancement of CollateralBlood Flow to Ischemic Myocardium by Vascular Endothelial Growth Factorin Dogs,” Circulation, 89(5):2183-2189 (May 1994), disclosessuccessfully inducing coronary angiogenesis (i.e., a 40% increase incollateral blood flow and an 89% increase in the numerical density ofintramyocardial distribution vessels) in dogs by administering 45 μg ofhuman recombinant VEGF/day for 5 days/week for 4 weeks to the distalleft circumflex artery (LCx) of dogs whose proximal LCx was constrictedbefore the first takeoff branch with an ameroid constrictor and whereina hydraulic balloon occluder was placed immediately distal to theencircling ameroid. In a similar study, Unger, et al., “Basic fibroblastgrowth factor enhances myocardial collateral flow in a canine model,”Am. J. Physiol., 266 (Heart Circ. Physiol. 35): H1588-H1595 (1994),disclose enhancing collateral blood flow (i.e., final collateral tonormal zone (CZ/NZ) blood flow ratios of 0.49 and 0.35 in the treatedand untreated groups, respectively) in dogs by administering a dailybolus of 110 μg of human recombinant basic FGF (the 155 residue form)for 9 days to the distal left circumflex artery (LCx) of dogs whoseproximal LCx was constricted before the first takeoff branch with anameroid constrictor and wherein a hydraulic balloon occluder was placedimmediately distal to the encircling ameroid. However, in the abovestudy, Unger was not able to show that his method or dosage inducedangiogenesis. Making any assessment based on collateral blood flow moredifficult, Unger also discloses that administration of basic FGF causesan acute vasodilatory effect, reducing blood pressure and increasingcoronary blood flow. Unger (1994) at page H1590, col. 2 and at pageH1592, col. 2.

In an earlier study, Unger, et al., “A model to assess interventions toimprove collateral bloodflow: continuous administration of agents intothe left coronary artery in dogs,” Cardiovascular Res., 27:785-791(1993), Unger discloses the continuous infusion for four (4) weeks of 30μg/hr recombinant acidic FGF (i.e., FGF-1) in the presence of 30 IU/hrheparin into the proximal end of the left circumflex artery (LCx) of adog after constricting the artery for four weeks with an ameroidconstrictor, followed by double ligation of the artery and insertion ofa catheter for infusing the FGF-1 into the proximal stub of the ligatedLCx. Unger (1993) at page 785. Notwithstanding that a total cumulativedose of 10 mg of acidic FGF was infused into the coronary artery of eachdog. Unger reported that in this model, “acidic FGF had nodemonstratable effect on collateral blood flow . . . . ” Unger (1993) atpage 785 (Abstract), and at page 790.

Harada, et al., “Basic Fibroblast Growth Factor Improves MyocardialFunction in Chronically Ischemic Porcine Hearts,” J. Clin. Invest.,94:623-630 (August 1994), disclose enhancing coronary blood flow andreduction in infarct size in a gradual coronary occlusion model inYorkshire pigs by extraluminal (periadvential) administration of 8 μg ofbasic FGF in the form of 4-5 capsules having 1 μg/capsule of basic FGFthat are positioned on the proximal left anterior descending artery(LAD) and both proximal and distal to an ameroid constrictor placed onthe proximal end of the left circumflex artery (LCx) before the firsttakeoff branch. Although an express object of Harada's experiment was to“alleviate chronic myocardial ischemia by stimulating angiogenesis”[Harada at page 628], Harada was not able to show angiogenesis.Moreover, Harada concluded that “[I]_(t) is not clear what is theoptimal dose of bFGF or the length or route of administration.” Haradaat page 629. Separately, Landau et al., “Intrapericardial basicfibroblast growth factor induces myocardial angiogenesis in a rabbitmodel of chronic ischemia,” Am. Heart Journal, 129:924-931 (1995),discloses that administering 180 ng/day of human recombinant basic FGF(154 residues) into the pericardial space of 2.0-4.3 kg rabbits for 7-28days, enhances new epicardial small-vessel growth, and that the effectis enhanced by left ventricular hypertrophy. The dosage of basic FGFutilized in Landau, when scaled to the size of a 70 kg man, wouldcorrespond to 2.9 μg/day for 7-28 days, or a total dose of basic FGF of20.3 μg-81.2 μg. Lopez et al., “Angiogenic potential of perivascularlydelivered aFGF in a porcine model of chronic myocardial ischemia,” Am.J. Physiol. 274 (Heart Circ. Physiol. 43): H930-H936 (1998), disclosesimproving myocardial flow and regional and global left ventricularfunction in Yorkshire pigs by perivascular delivery of 14 μg of arecombinant human aFGF mutein (i.e., Ser-117 aFGF, wherein Ser replacesCys) that is diffusely distributed in a porous ethylene vinyl acetate(EVA) polymer that is secured with sutures over the proximal leftcircumflex artery. Lopez reports that the perivascularly delivered aFGFimproved blood flow in the compromised region of the heart in animalsboth “at rest” and “during rapid pacing.” Lopez at page H934, col. 2.However, Lopez was unable to directly attribute the increased blood flowto angiogenesis, citing other possible sources, such as “vasodilation”or “improvements in vascular circulation.”

Finally, U.S. Pat. No. 4,296,100, which issued to Franco on Oct. 20,1981, discloses a method for treating a myocardial infarction inpatients by administering 10 mg to 1 g of 90% pure bovine FGF (pituitaryextract) per 100 g of heart tissue as a one-time treatment immediatelyfollowing infarct. According to Franco, “[a]_(t) least 10 micrograms/100grams heart is used to achieve the effect desired.” Franco at col. 1,lines 62-64. Franco discloses that the FGF is administered to the heartby a variety of modes, including direct injections into the heart,intravenous injection, subcutaneous injection, intramuscular injectionand oral ingestion. Franco at col. 2, lines 63-69. Franco also disclosesthat his method was able to reduce infarct size (area of scarring or ofpermanent damage) to one quarter of that in the control. Franco at TableIII. According to Franco, the function of the FGF was to “increase bloodflow for a sustained period of time after myocardial infarction.” Francoat col. 1, lines 42-43. However, the acute affect of any FGFadministration is vasodilation, which inherently increases coronaryblood flow. Franco expressly discloses that a histological study “didnot show any significant increase in capillary areas in the hearts” as aresult of such treatment with 10 μg to 1 g of FGF per 100 g of heart.Franco at col. 4, lines 13-17. Moreover, Franco did not address theissue of whether administering such large doses of FGF would haveangiogenic effects in any undiscovered tumors in the body.

Thus, it is an object of the present invention to provide a dosage of anangiogenic agent and a method of administering one or more dosages ofthe angiogenic agent to a patient in an amount that is effective toinduce angiogenesis to an area of the heart in need of angiogenesis. Itis a further object of this application to provide a dosage and a methodfor delivering an angiogenic agent that would provide for a therapeuticeffect, including angiogenesis at the target site, while reducing therisk of inducing angiogenesis at an unwanted site elsewhere in the body.

The above-described references and all other references cited herein areexpressly incorporated herein in their entirety.

SUMMARY OF THE INVENTION

The Applicants have unexpectedly discovered that certain dosages of anangiogenic agent, when injected into the myocardium downstream from acoronary occlusion, provided that portion of the myocardium with atherapeutic response as reflected by an increase in resting regionalperfusion, an improvement in regional cardiac function, and increasedvascularity. In particular, the Applicants discovered that a unit dose(i.e., from about 5 ng/dose to less than 135,000 ng/dose) of anangiogenic agent, when administered directly into the myocardium as asingle injection or as a series of injections in the area of need,induced coronary angiogenesis in the myocardium in the area ofadministration but became sufficiently diluted elsewhere in the body tominimize any risk of inducing angiogenesis. When the unit dose ofangiogenic agent of the present invention is administered as a series ofinjections, the series of injections are administered as a singleprocedure on the same day or as a series of injections on successive oralternating days as needed. However, the cumulative amount of the dosageof the angiogenic agent that is administered is typically from about 5ng to less than 135,000 ng (135 μg), more typically from 5 ng to 67,500ng (67.5 μg). Thus, in one aspect, the present invention is directed toa unit dose pharmaceutical composition (“pharmaceutical composition”)comprising from about 5 ng to less than 135,000 ng (preferably from 5 ngto 67,500 ng) of an angiogenic agent in a pharmaceutically acceptablecarrier. In another aspect, the present invention is directed to a unitdose pharmaceutical composition (“unit dose composition”) comprisingfrom about 5 ng to less than 135,000 ng (preferably from 5 ng to 67,500ng) of an angiogenic agent in a pharmaceutically acceptable carrier.

In yet another aspect, the present invention is directed to method forinducing angiogenesis, or increasing regional perfusion, or increasingcardiac function, or increasing vascular density in the myocardium of apatient in need of such treatment, comprising injecting a unit dosage ofan angiogenic agent, as a single injection or as a series of injections,directly into an area of myocardium in need of such angiogenesis, orincreased regional perfusion, or increased cardiac function, orincreased vascular density, respectively. It is also within the scope ofthe above-described method that the step of injecting the unit dosage beperformed as a single injection, or preferably as a series of injectionson the same day. Regardless of whether the above method is performedusing a single injection or a series of injections, the cumulativeamount of the angiogenic agent that is injected into the area ofmyocardium in need of angiogenesis during the one or more injections isfrom about 5 ng to less than 135,000 ng (135 μg).

It is also appropriate to express the unit dose of the present inventionas μg of angiogenic agent per kilogram (kg) of patient weight. When soexpressed, a dose of angiogenic agent for intramyocardial (IMc)injection in accordance with the present invention ranges from about0.06 μg angiogenic agent to about 10.0 μg angiogenic agent per kg ofpatient weight (hereinafter “μg/kg”). More typically, the dose ofangiogenic agent ranges from 0.06 μg/kg to 6.0 μg/kg. However, becausethe angiogenic agent is being injected directly into the myocardium ofthe patient in the method of the present invention, the typicaldilutional effects on dosage associated with patient body weight areminimal, particularly when compared to systemic or intracoronoryadministration of the same amount of angiogenic agent.

Two diseases where angiogenesis increased regional perfusion, andincreased coronary vascularity are desirable are coronary artery disease(CAD) and myocardial infarction (MI). Thus, in another aspect, thepresent invention is also directed to a method for treating a patientfor coronary artery disease (CAD) comprising injecting a unit dosage ofan angiogenic agent, as a single injection or as a series of injections,directly into a portion of the myocardium manifesting symptoms of CAD,the unit dosage containing an amount of the angiogenic agent (about 5 ngto less than 135,000 ng) that is effective to induce angiogenesis, orincrease regional perfusion, or increase myocardial function by DSE atpeak stress, or increase vascularity in the area of myocardiummanifesting said symptoms. In another aspect, the present invention isdirected to a method for treating a patient for a myocardial infarction(MI) comprising injecting a unit dosage of an angiogenic agent, as asingle injection or as a series of injections, directly into an area ofmyocardium manifesting symptoms of coronary insufficiency as a result ofsaid MI. In the above-described method, the unit dose of angiogenicagent that is effective in treating said myocardial infarction is about5 ng to less than 135,000 ng of angiogenic agent/unit dose, moretypically 5 ng to 67,500 ng of angiogenic agent/unit dose.

Although the unit dose is typically injected into the myocardium on asingle day, it is within the scope of the present invention that thestep of injecting the unit dose of angiogenic agent be performed orrepeated on successive or alternating days or weekly or monthly asneeded. Regardless of whether the above method is repeated, thecumulative amount of the angiogenic agent that is injected into the areaof myocardium in need of angiogenesis during any single intervention isfrom about 5 ng to less than 135,000 ng of said angiogenic agent.

A suitable angiogenic agent for use in the unit dose or pharmaceuticalcomposition of the present invention is selected from the groupconsisting of a platelet-derived growth factor (PDGF), vascularendothelial growth factor (VEGF-A), VEGF-B, VEGF-D, transforming growthfactor-β (TGF-β1), fibroblast growth factor (FGF), or an angiogenicallyactive fragment or mutein thereof. Preferably, the angiogenic agent isVEGF-A, VEGF-D, an FGF, or an angiogenically active fragment or muteinthereof. More preferably, the angiogenic agent is an FGF, such as FGF-1,FGF-2 or FGF-5, or an angiogenically active fragment or mutein thereof.Most preferably, the angiogenic agent is FGF-2, or an angiogenicallyactive fragment or mutein thereof.

The duration of the therapeutic effects provided by the method of thepresent invention is wholly unexpected. In particular, when a singleunit dose of 0.06 μg/kg (1,350 ng total dose) of recombinant bovineFGF-2 (SEQ ID NO: 2) was administered by injection directly to themyocardium of a miniswine having 90% occlusion of the left circumflexcoronary artery (i.e., providing a model of a hibernating myocardium),improvements were seen in the resting mean blood flow (MBF), the wallmotion score index (WMSI), vascular perfusion, myocardial function, andvascular density in the hibernating myocardial tissue, which continuedfor as far out as the six (6) month measurement period. By way ofexample, the resting MBF increased from a baseline of 64±0.04% ofnon-ischemic septal flow to 71±0.05%, p<0.05 vs baseline at one monthpost-treatment and to 76±0.06, p<0.05 vs baseline at three monthspost-treatment. At six months post-treatment, the resting MBF increasedfrom 61.3±4.4% of non-ischemic septal flow at baseline to 82.8±3.1%. Inanother test that is accepted as a measure of contractile reserve, thewall motion score index (WMSI) measured at rest for the LCx region(after 90% stenosis of the LCx) improved from 2.4±0.2 to 2.2±0.2 (p=0.08vs baseline) at 6 months post-treatment. Similarly, the wall motionscore index (WMSI) measured at peak stress for the LCx region (after 90%stenosis of the LCx) improved significantly, decreasing from 2.2±0.4 to1.8±0.3 (p=0.05 vs baseline) at 6 months post-treatment. These decreasesin the wall motion score index are consistent with a reduction inischemia. In contrast, the patients (miniswine) that were treated withthe vehicle for the angiogenic agent exhibited no significant change inresting MBF, and no significant change in their resting or stress WMSIat any time during the six-month post-treatment period.

In addition, after intramyocardial (IMc) injection of a single unit doseof FGF-2 (0.06 μg/kg, i.e., 1.35 μg) in the above-described pig model ofa hibernating myocardium, normalized perfusion, which is reported as %change in perfusion, continued to increase throughout the measurementperiod from 18% to 38% at 3 and 6 months, respectively, compared toincreases of 6% and 13% at 3 and 6 months, respectively for saline. SeeFIG. 4. When three different embodiments of the unit dose of the presentinvention, i.e., a unit dose containing 0.06 μg/kg (1.35 μg) of rFGF-2(SEQ ID NO: 2) “low” dose; a unit dose containing 0.6 μg/kg (13.5 μg) ofrFGF-2 (SEQ ID NO: 2) “mid” dose; a unit dose containing 6.0 μg/kg (135μg) of rFGF-2 (SEQ ID NO: 2) “high” dose, were injected IMc into the pigmodel of the hibernating myocardium (90% occlusion of the LCx) andcompared to intracoronary (IC) injection of the “mid” dose in theameroid model (100% occlusion of the LCx), all IMc injections produced anormalized perfusion at 3 months that was superior to that produced byIC injection. FIGS. 7 and 8. Suprisingly, the mid dose resulted in 10%greater normalized perfusion than that produced by either the low doseor the high dose at three months post-dosing. FIG. 7.

Myocardial function, as measured by a dolbutamine stress echocardiogram(DSE), showed statistically significant increases in myocardial function(lower number) at 3 and 6 months after injection of each of threedifferent unit doses of the present invention (low, medium and high)into the pig model of the hibernating myocardium, compared to injectionof placebo and IC injection of the “mid” dose in the ameroid pig model.See FIGS. 5 and 11. Injection of a single unit dose of FGF-2 (1.35 μg)IMc into the pig model of the hibernating myocardium produced astatistically significant (p<0.05) increase in vascularity of thetreated hibernating myocardium at 6 months post dosing, as measured bythe number of capillaries (44,000) in a fixed volume of the FGF-2treated ischemic myocardium versus the number of capillaries (17,000) inthe same fixed volume of saline treated myocardium. See FIG. 6.

Finally, Western blot analysis of myocardial tissue from the ischemicregions of the myocardium treated with FGF-2 IC or IMc, indicated thatthere was a significant upregulation of VEGF (measured as VEGF₁₆₅) andFGF-2, which was detectable even at the end of the observation period(i.e., 3 months after injection) versus those regions treated withvehicle alone. See FIG. 10. Surprisingly, the FGF-2 treated ischemiccells were producing statistically significant amounts of both VEGF andFGF-2 3 months after treatment. More suprisingly, the highestconcentration (greater than 290 pg/ml) of intracellular FGF-2 wasobserved in the ischemic myocardial tissue that was treated 3 monthsearlier with the “mid” dose (0.6 μg/kg, i.e., 13.5 μg) of FGF-2 of SEQID NO: 2 IMc. See FIG. 10. In contrast, the “high” dose (6.0 μg/kg,i.e., 135 μg) of FGF-2, while providing for comparable intracellularconcentrations of VEGF (about 100 pg/ml), only provided for aconcentration of intracellular FGF-2 that was about 165 pg/ml. See FIG.10. Thus, the “mid” dose of FGF-2, when administered IMc not onlystimulated the treated ischemic myocardial cells to produce endogenousVEGF and FGF-2 for three months after treatment, but also stimulatedthose cells to produce almost twice the concentration of FGF-2 producedby the cells treated with the “high” dose. Given this and the other dataprovided herein, we would expect production of an unexpectedly superioramount of intracellular FGF-2 to be stimulated by IMc injection of adose of FGF-2, ranging from about 0.3 μg/kg (or 6.75 μg) to about 3.0μg/kg (or 67.5 μg). (The data at six months is not yet available.) Thepresence of both VEGF and FGF-2 suggests a mechanism by which occurincreases in perfusion, myocardial function and vascular permeability.Thus, in another aspect, the present invention is directed to a methodfor increasing the intracellular concentration of VEGF and FGF-2 inischemic myocardial tissue, comprising injecting the ischemic myocardialtissue with a unit dose of an angiogenic agent. Preferably, theangiogenic agent is an FGF; more preferably, FGF-2.

The method of the present invention was found to improve cardiacfunction for up to 6 months after treatment when compared to variouscontrol groups. Specifically, the % change in peak-stress normalizedregional function score was found to decrease, indicating improvedcardiac function, for the IMc administered groups at 3 and 6 months posttreatment, and that it increased, indicating decreasing function, forthe IC and placebo groups. FIG. 11. In addition, the greatest decreasein normalized function scores surprisingly occurred with the “low” dosegroup, and even more surprisingly showed, by the decreasing functionscore, that regional myocardial function continued to improve up to 6months after treatment with the low dose. FIG. 11.

Many of the angiogenic agents, such as acidic FGF (aFGF or FGF-1), basicFGF (bFGF or FGF-2), and VEGF are glycosoaminoglycan binding proteins.The presence of a glycosoaminoglycan (also known as a “proteoglycan” ora “mucopolysaccharide”) optimizes the angiogenic activity and AUC ofthese angiogenic agents. As a result, the unit dosages of FGF-1, FGF-2,VEGF-A, VEGF-B, VEGF-D or the angiogenic fragments and muteins thereof,optionally are administered within 20 minutes of the IV administrationof a glycosoaminoglycan, such as a heparin. However, in our experience,the presence of an aminoglycan was not needed for efficacy when a unitdose of an angiogenic agent, e.g., FGF-2, was administered IMc inaccordance with the method of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of the mean recombinant bovine FGF-2 plasmaconcentration versus time (hours) for six different doses of rFGF-2administered by IC infusion in humans over a 20-minute period. The sixdoses of rFGF-2 in FIG. 1 are 0.33 μg/kg, 0.65 μg/kg, 2 μg/kg, 6 μg/kg,12 μg/kg, and 24 μg/kg of lean body mass (LBM).

FIG. 2 is a plot of each individual patient's rFGF-2 area under thecurve (AUC) in pg·hr/ml for FIG. 1 for the six doses of rFGF-2, andshows the dose linearity of systemic rFGF-2 exposure following ICinfusion.

FIG. 3 is a plot of individual human patient rFGF-2 dose normalized AUCsas a function of the time of heparin administration in “minutes prior torFGF-2 infusion” and shows the influence of timing of heparinadministration on rFGF-2 AUC. The rFGF-2 was recombinant bovine FGF-2.

FIG. 4 is a bar graph comparing the normalized myocardial perfusion(reported as % change from baseline) in the pig model of the hibernatingmyocardium, as measured by positron emission tomography (PET), at 3 and6 months following: sham administration; saline; and a unit dosecontaining 1.35 μg of rFGF-2 (SEQ ID NO: 2).

FIG. 5 is a bar graph comparing myocardial function by dolbutaminestress echocardiogram in the pig model of the hibernating myocardium atbaseline, 3 months and 6 months following sham administration; saline;and a unit dose containing 1.35 μg of rFGF-2 (SEQ ID NO: 2).

FIG. 6 is a bar graph comparing capillary density (# of vessels) in theischemic myocardial tissue (downstream from the 90% occlusion in theLCx) in the pig model of the hibernating myocardium at 6 monthsfollowing sham administration; saline; and a unit dose containing 1.35μg of rFGF-2 (SEQ ID NO: 2).

FIG. 7 is a bar graph, comparing the normalized myocardial perfusion(reported as % change from baseline) in the pig model of the hibernatingmyocardium, as measured by positron emission tomography (PET), at 3months following: administering saline (placebo); a unit dose containing0.06 μg/kg (1.35 μg) of rFGF-2 (SEQ ID NO: 2) “low”; a unit dosecontaining 0.6 μg/kg (13.5 μg) of rFGF-2 (SEQ ID NO: 2) “mid”; a unitdose containing 6.0 μg/kg (135 μg) of rFGF-2 (SEQ ID NO: 2) “high”. Thebar graph shows that the greatest % change in normalized perfusion(i.e., a 27.5% increase) occurred for the “mid” dose, with the “low” and“high” doses showing comparable changes, 17.5% and 17%, respectively.The data in FIG. 7 is the result of two separate experiments (light barsand dark bars) with the placebo designated as “uld” (ultra-low dose)being the placebo for the “low” dose, shown as the light colored bars.

FIG. 8 is a bar graph, comparing the % change in normalized myocardialperfusion (as measured by PET) in the pig model of the ameroid (100%occlusion of the LCx) myocardium at 1 month and 3 months afterintracoronary (IC) infusion of 0.6 μg/kg rFGF-2, versus the % change innormalized myocardial perfusion in the pig model of the hibernatingmyocardium (90% occlusion of the LCx) at 1 month and 3 months afterintramyocardial (IMc) injection of the following: saline (placebo); aunit dose containing 0.6 μg/kg (13.5 μg) of rFGF-2 (SEQ ID NO: 2) “mid”;a unit dose containing 6.0 μg/kg (135 μg) of rFGF-2 (SEQ ID NO: 2)“high”. The bar graph shows that the greatest % increase in normalizedperfusion occurred for the “mid” dose of rFGF-2 IMc at 3 months posttreatment. The “high” dose unexpectedly showed a lower increase innormalized perfusion than was achieved for the “mid” dose.

FIG. 9 is a bar graph comparing vascular density (average vessel numberin a designated volume of treated myocardium) for the pig model of thehibernating myocardium treated with 0.6 μg/kg (“mid” dose) or 6.0 μg/kg(“high” dose) rFGF-2 (SEQ ID NO: 2) IMc versus the ameroid pig model(100% occlusion of the LCx) treated with 6.0 μg/kg rFGF-2 (SEQ ID NO: 2)IC, versus treatment with saline IMc (placebo). The results show thatthe greatest increase in vascular density was produced by the “mid” dose(0.6 μg/kg or 13.5 μg rFGF-2) that was administered IMc.

FIG. 10 is a bar graph comparing the intracellular concentrations(pg/ml) of VEGF (measured as VEGF₁₆₅) and FGF-2 in ischemic myocardialcells 3 months after treatment with 0.6 μg/kg (13.5 μg) FGF-2 of SEQ IDNO: 2 IC in the ameroid pig model (100% occlusion of the LCx), or withvehicle or 0.6 μg/kg (13.5 μg) FGF-2 of SEQ ID NO: 2 (“mid” dose) or 6.0μg/kg (135 μg) FGF-2 of SEQ ID NO: 2 (“high” dose) in the pig model ofthe hibernating myocardium (90% occlusion of the LCx). Surprisingly, theFGF-2 treated ischemic cells were producing statistically significantamounts of both VEGF and FGF-2 up to 3 months after treatment. Moresurprisingly, the highest concentrations of intracellular FGF-2 wereinduced by those cells treated with the “mid” dose IMc.

FIG. 11 is a bar graph comparing the % change in peak-stress normalizedregional function score by DSE at 3 months and 6 months after treatmentwith placebo, or with the “mid” dose (0.6 μg/kg (13.5 μg)) of FGF-2 (SEQID NO: 2) IC in the ameroid pig model, or with the “low” dose (0.06μg/kg (1.35 μg)) of FGF-2 (SEQ ID NO: 2) IMc, or with the “mid” dose(0.6 μg/kg (13.5 μg)) of FGF-2 (SEQ ID NO: 2) IMc, or with the “high”dose (6.0 μg/kg (135 μg)) of FGF-2 (SEQ ID NO: 2) IMc in the pig modelof the hibernating myocardium. FIG. 11 shows that the % change inpeak-stress normalized regional function score decreased, indicatingbetter function, for the IMc administered groups at 3 and 6 months posttreatment, and that it increased, indicating decreasing function, forthe IC and placebo groups. In addition, the greatest decrease innormalized function scores occurred with the “low” dose group andsurprisingly showed, by the decreasing function score, that functioncontinued to improve up to 6 months after treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon human clinical trials of patientsmanifesting symptoms of coronary artery disease (CAD) and uponcomparative testing of the effects produced by various modes ofadministering a recombinant angiogenic agent in two porcine models ofcoronary insufficiency. The porcine heart is considered to be aparticularly relevant model of the human heart because of itsresemblance to the human coronary circulation and its paucity of naturalcollateral circulation. See Battler et al., “Intracoronary Injection ofBasic Fibroblast Growth Factor Enhances Angiogenesis in Infarcted SwineMyocardium,” JACC, 22(7): 2001-6 (December 1993) at page 2002, col. 1(“the canine model of myocardial ischemia has been criticized because ofthe abundance of naturally occurring collateral circulation, as opposedto the porcine model, which ‘excels’ in its relative paucity of naturalcollateral circulation and its resemblance to the human coronarycirculation.”). One of the animal models employed was the porcine modelof a hibernating myocardium. This model was created by surgicallyplacing a hydraulic occluder on the proximal end of the left circumflexcoronary artery (LCx). Distal to the occluder, there was placed anembedded flow probe which was continuously monitored the occlusion tomaintain it at 90%. The hibernating cardiac model is a particularlyrelevant model of coronary artery disease. Heart muscle may beclassified as healthy, hibernating or dead. Dead tissue is not dead butscarred, non-contracting, and no longer capable of contracting even ifit were supplied adequately with blood. Hibernating tissue isnon-contracting muscle tissue, but is capable of contracting, should itbe adequately resupplied with blood. Healthy heart tissue is identifiedby strong electrical signals in combination with strong displacement.“Dead or diseased heart tissue is identified by weak electrical signalsin combination with dysfunctional displacement, i.e., displacement in adirection opposite that of healthy tissue. Ischemic, or hibernating orstunned heart tissue is identified by strong electrical signals incombination with impaired displacement.” See U.S. Pat. No. 5,897,529(Ponzi), which issued Apr. 27, 1999. The diagnosis of hibernating tissueis critical because it is widely believed that once the occlusion iseliminated, there is an immediate return of normal function. See U.S.Pat. No. 5,743,266 (Levene), which issued Apr. 28, 1998. Thus, thehibernating model of the myocardium is similar to what occurs in a humanpatient having coronary artery disease (CAD) and/or chronic anginawherein one or more coronary vessels are partially occluded.

In the porcine ameroid model, an ameroid constrictor, which is adonut-like band or ring that has a hygroscopic material on its innerface, was placed around the proximal end of the LCx in a pig. Thehygroscopic material gradually swells and provides 100% occlusion of theartery in 10 days to 3 weeks. Unlike the hibernating model wherein thepercentage of occlusion is hydraulically controllable, consistent andreliable, the ameroid model lacks a consistent control. Also, thecomplete occlusion in the ameroid model leads to infarction andextensive spontaneous collateral formation, which causes mean blood flowin the resting state to return back to normal, making it more difficultto attribute a particular amount of collateral formation to exogenouslyadministered angiogenic agent. Thus, the ameroid model is not asstringent a model as the hibernating myocardium model. Moreover, the100% occlusion that is provided by the ameroid model makes the ameroidmodel more analogous to a myocardial infarction, where there is 100%occlusion of one or more coronary arteries.

Using the above described models, the Applicants discovered that a dose(i.e., from about 5 ng/dose to less than 135,000 ng/dose) of anangiogenic agent (i.e., a unit dose) when administered as a singleinjection or as a series of injections directly into an ischemic area ofthe myocardium, induced coronary angiogenesis in the myocardium in thearea of administration, but became sufficiently diluted elsewhere in thebody to minimize any risk of inducing angiogenesis. More typically, thecumulative amount of angiogenic agent that is administered to themyocardium of a patient is from 5 ng to 67,500 ng of angiogenic agent.Thus, in one aspect, the present invention is directed to a unit dosepharmaceutical composition (“unit dose”) comprising from about 5 ng toless than 135,000 ng (preferably from 5 ng to 67,500 ng) of anangiogenic agent in a pharmaceutically acceptable carrier.

By the term “angiogenesis” or “coronary angiogenesis,” as used herein,is meant the formation of new blood vessels, ranging in size fromcapillaries to arterioles which act as collaterals in coronarycirculation. In the present invention, angiogenesis was measured usingone or more art-accepted indicators that assessed changes in myocardialperfusion, function as measured by a dolbutamine stress echocardiogram,and capillary density.

By the term “angiogenic agent,” as used herein, is meant a memberselected from the group PDGF, VEGF-A, VEGF-B, VEGF-D, TGF-β1, FGF, or anangiogenically active mutein or fragment thereof. Preferably, theangiogenic agent is VEGF-A, VEGF-D or an FGF or an angiogenically activefragment or mutein thereof. More preferably, the angiogenic agent is anFGF. Most preferably, the angiogenic agent is FGF-2, or anangiogenically active fragment or mutein thereof.

By the phrase “angiogenically active fragment” is meant a protein orpolypeptide fragment of an angiogenic agent that exhibits at least 80%of the angiogenic activity of the parent molecule from which it wasderived.

By the phrase “angiogenically active mutein,” as used herein, is meantan isolated and purified recombinant protein or polypeptide that has 65%sequence identity (homology) to any naturally occurring angiogenic agentselected from the group PDGF, VEGF-A, VEGF-B, VEGF-D, TGF-β1 and FGF, asdetermined by the Smith-Waterman homology search algorithm (Meth. Mol.Biol. 70:173-187 (1997)) as implemented in the MSPRCH program (OxfordMolecular) using an affine gap search with the following searchparameters: gap open penalty of 12, and gap extension penalty of 1, andthat retains at least 80% of the angiogenic activity of the naturallyoccurring angiogenic agent with which it has at least 65% sequenceidentity. Preferably, the angiogenically active mutein has at least 75%,more preferably at least 85%, and most preferably, at least 90% sequenceidentity to the naturally occurring angiogenic agent. Other well-knownand routinely used homology/identity scanning algorithm programs includePearson and Lipman, PNAS USA, 85:2444-2448 (1988); Lipman and Pearson,Science, 222:1435 (1985); Devereaux et al., Nuc. Acids Res., 12:387-395(1984); or the BLASTP, BLASTN or BLASTX algorithms of Altschul, et al.,Mol. Biol., 215:403-410 (1990). Computerized programs using thesealgorithms are also available and include, but are not limited to: GAP,BESTFIT, BLAST, FASTA and TFASTA, which are commercially available fromthe Genetics Computing Group (GCG) package, Version 8, Madison Wis.,USA; and CLUSTAL in the PC/Gene program by Intellegenetics, MountainView Calif. Preferably, the percentage of sequence identity isdetermined by using the default parameters determined by the program.

The phrase “sequence identity,” as used herein, is intended to refer tothe percentage of the same amino acids that are found similarlypositioned within the mutein sequence when a specified, contiguoussegment of the amino acid sequence of the mutein is aligned and comparedto the amino acid sequence of the naturally occurring angiogenic agent.

When considering the percentage of amino acid sequence identity in themutein, some amino acid residue positions may differ from the referenceprotein as a result of conservative amino acid substitutions, which donot affect the properties of the protein or protein function. In theseinstances, the percentage of sequence identity may be adjusted upwardsto account for the similarity in conservatively substituted amino acids.Such adjustments are well known in the art. See, e.g., Meyers andMiller, “Computer Applic. Bio. Sci., 4:11-17(1988).

To prepare an “angiogenically active mutein” of an angiogenic agent ofthe present invention, one uses standard techniques for site-directedmutagenesis, as known in the art and/or as taught in Gilman, et al.,Gene, 8:81 (1979) or Roberts, et al., Nature, 328:731 (1987). Using oneof the site-directed mutagenesis techniques, one or more point mutationswould introduce one or more conservative amino acid substitutions or aninternal deletion. Conservative amino acid substitutions are those thatpreserve the general charge, hydrophobicity/hydrophilicity, and/orsteric bulk of the amino acid being substituted. By way of example,substitutions between the following groups are conservative: Gly/Ala,Val/Ile/Leu, Lys/Arg, Asn/Gln, Glu/Asp, Ser/Cys/Thr, and Phe/Trp/Tyr.Significant (up to 35%) variation from the sequence of the naturallyoccurring angiogenic agent is permitted as long as the resulting proteinor polypeptide retains angiogenic activity within the limits specifiedabove.

Cysteine-depleted muteins are muteins within the scope of the presentinvention. These muteins are constructed using site-directed mutagenesisas described above, or according to the method described in U.S. Pat.No. 4,959,314 (“the '314 patent”), entitled “Cysteine-Depleted Muteinsof Biologically Active Proteins.” The '314 patent discloses how todetermine biological activity and the effect of the substitution.Cysteine depletion is particularly useful in proteins having two or morecysteines that are not involved in disulfide formation.

One of the angiogenic agents in the pharmaceutical composition and unitdose of the present invention is PDGF. PDGF is a family of three dimericangiogenically active proteins, PDGF-AA, PGDF-AB and PGDF-BB, whereinseparate genes encode the A-chain and the B-chain, respectively. ThePDGF receptor type-alpha (PDGFR-o) binds both the A- or B-chain of thePDF dimers with high affinities, whereas the PDGF receptor type-β(PDGFR-β) only binds the B-chain. All of the PDGFs are angiogenicallyactive in vivo. See Carmeliet, et al., “Vascular development anddisorders: Molecular analysis and pathogenic insights,” KidneyInternatl., 53:1519-1549 (1998); Risau et al., “Platelet-derived growthfactor is angiogenic in vivo,” Growth Factors, 7:261-266 (1992);Martins, et al., “The role of PDGF-BB on the development of thecollateral circulation after acute arterial occlusion,” 10:299-306(1994); and Brown et al., “Platelet-derived growth factor BB inducesfunctional vascular anastomoses in vivo,” PNAS USA, 92:5920-5924 (1995),which are hereby incorporated herein by reference in their entirety. Allother references cited herein, either before or after, are expresslyincorporated herein by reference in their entirety. The DNA sequence andthe amino acid sequence for the 211 amino acid residue human PDGFA-chain precursor are known in the art. See U.S. Pat. No. 5,219,759,entitled “Recombinant DNA Encoding PDGF A-chain Polypeptide andExpression Vectors,” which issued on Jun. 15, 1993 to Hedlin et al.(“the '759 patent”) at FIG. 1. The amino acid sequence for the 125residue mature PDGF A-chain corresponds to residues 87-211 of FIG. 1 ofthe '759 patent. The '759 patent at FIG. 2 also discloses a cDNA and thededuced amino acid sequence of a variant PDGF A-chain precursor protein,having only 196 amino acid residues, wherein the 110 residue mature PDGFA-chain corresponds to residues 87-196 of the deduced sequence. Thefirst 107 residues of the mature PDGF A-chains (i.e., residues 87-193)are identical. See the '759 patent at FIGS. 1 and 2. Thus, the remainingresidues, i.e., residues 108-125 of a mature PDGF A-chain are notcritical for activity and may be conservatively substituted withoutadverse effect. In addition, as the 110 residue variant PDGF A-chain ofFIG. 2 of the '759 patent shows, the residues beyond residue 110 of the125 residue mature PDGF are not necessary for activity, and may bedeleted to provide a series of deletion muteins that also would beexpected to be functional in the present invention. Another referencediscloses that the mature A-chain has 104 amino acids. See U.S. Pat. No.5,512,545, entitled “PDGF-B Analogues,” which issued on Apr. 30, 1996 inthe name of Brown et al. (“the '545 patent”), at col. 2, lines 40-44.Thus, the '545 patent suggests that any residues beyond the first 104 ofmature PDGF-A are not critical to PDGF-A activity.

Likewise, the DNA sequence and the deduced amino acid sequence for humanPDGF B-chain are known in the art and disclosed in FIGS. 2 and 3,respectively, in the '545 patent. The mature PDGF-A and PDGF-B chainsshow 60% homology and the 8 cysteine residues in each chain areconserved. Although PDGF B-chain may have the full complement of 160amino acids shown in FIG. 2 and SEQ ID NO: 1 of the '545 patent, thelast 51 residues may be removed without loss of activity. The resultingcarboxy-truncated PDGF B-chain has 109 residues (i.e., residues 1-109 ofSEQ ID NO: 1 and FIG. 3 of the '545 patent) and contains the bindingregion, which occurs between residues 25 (Ile) and 37 (Phe). If the PDGFB-chain is expressed in yeast, it is desirable to replace the Arg atresidue positions 28 or 32 or both with a non-basic, neutral residue toprevent cleavage by the yeast cells. Methods, vectors, and cells forexpressing the PDGF A-chain and B-chain, and for combining theseA-chains and B-chains to make the three isoforms of PDGF are well knownin the art. See U.S. Pat. Nos. 5,605,816 and 5,512,545 as cited above.

Another angiogenic agent that is an active agent in the pharmaceuticalcomposition and unit dose of the present invention is VEGF. VEGF is abasic, homodimeric protein having a molecular weight of about 45,000Daltons (45 kD) that has four homologues, designated as VEGF (orVEGF-A), VEGF-B, VEGF-C and VEGF-D. For clarity herein, the first memberof the family, VEGF, will be referred to herein as VEGF-A. The VEGFfamily of proteins is characterized by having a highly conserved centralregion, characterized by the invariant presence in homologous positionsof 15 cysteine residues, 8 of which are involved in intra- andintermolecular disulfide bonding. See Ferrara, et al., “The Biology ofVascular Endothelial Growth Factor,” Endocrine Reviews, 18(1):4-25(1997) at FIG. 4. As a result, the four VEGF homologues have a similarshape (tertiary structure) and are capable of spontaneously formingheterodimers when coexpressed. Accordingly, deletion muteins at the N-and C-terminal ends of the VEGFs that retain the internal cysteineswould be expected to retain their shape, form dimers and be biologicallyactive. The homologous positioning of 8 of the 15 conserved cysteineresidues of VEGF correspond to the 8 conserved cysteine residues of thePDGF family as comparatively shown in e.g., WO 98/02543 at FIG. 3; andKeck, et al., “Vascular Permeability Factor, an Endothelial Cell MitogenRelated to PDGF,” Science 246:1309-1312 (1989) at page 1311, col. 2 andFIG. 4.

Human VEGF-A exists in four isoforms, having 121, 165, 189 and 206 aminoacids, respectively. These four isoforms are designated as VEGF-A₁₂₁,VEGF-A₁₆₅, VEGF-A₁₈₉, and VEGF-A₂₀₆, respectively. See Ferrara, et al.,“The Biology of Vascular Endothelial Growth Factor,” Endocrine Reviews,18(1):4-25 (1997) at page 5. The human VEGF-A gene is organized intoeight (8) exons separated by seven (7) introns and its coding regionspans 14 kb. Id. Alternative exon splicing of the single VEGF-A geneaccounts for all of the heterogeneity. VEGF-A₁₆₅ lacks the residuesencoded by exon 6, while VEGF-A₁₂₁ lacks the residues encoded by exons 6and 7. Id. The three shorter isoforms of VEGF-A are based upon VEGF-A₂₀₆and reflect splice variations that occur in the carboxy half of themolecule. However, the last six amino acids (exon 8) of the carboxyterminus are conserved in all four splice variants.

The cDNA sequence that encodes human VEGF-A₁₂₁, and the correspondingamino acid sequence are well known in the art. See Leung, et al.,“Vascular endothelial growth factor is a secreted angiogenic mitogen,”Science 246:1306-1309 (1989) at FIG. 2B as described at page 1307, col.3. The cDNA and deduced amino acid sequence for human VEGF-A₁₆₅ are alsowell known in the art. See Leung, et al., “Vascular endothelial growthfactor is a secreted angiogenic mitogen,” Science 246:1306-1309 (1989)at page 1307 and FIG. 2B. Likewise, the cDNA and deduced amino acidsequence for human VEGF-A₁₈₉ have been well known in the art since 1991.See Keck, et al., “Vascular Permeability Factor, an Endothelial CellMitigen Related to PDGF,” Science, 246:1309-1312 (1989); see alsoTischer et al., “The human genefor vascular endothelial growthfactor,”J. Biol. Sci., 266:11947-11954 (1991). Finally, the cDNA and deducedamino acid sequence for human VEGF-A₂₀₆ are also well known in the art.See Houck, et al., “The vascular endothelial growth factor family:identification of a fourth molecular species and characterization ofalternative splicing of RNA,” Mol. Endocrinol. 5:1806-1814 (1991) atFIG. 2A.

An overlapping comparison of the amino acid sequences of the four splicevariants (isoforms) of VEGF-A is shown in Ferrara, et al., “Molecularand Biological Properties of the Vascular Endothelial Growth FactorFamily of Proteins,” Endocrine Reviews 13(1):18-32 (1992) at page 21,FIG. 1. The shortest isoform, VEGF-A₁₂₁, which is freely soluble in theextracellular milieu, is slightly acidic due to the absence of most ofthe carboxy terminus (i.e., exons 6 and 7) which are rich in basic aminoacid residues. The longer isoforms, VEGF-A₁₆₅, VEGF-A₁₈₉, and VEGF-A₂₀₆,are less soluble, and thus, less diffusible, than VEGF-A₁₂₁, but exhibitboth a mitogenic activity and a binding affinity for a heparin-richmatrix that increases with increasing length at the carboxy terminus. Byway of example, VEGF-A₁₆₅ is more than 100-fold more mitogenic thanVEGF-A₂₁. See Carmeliet et al., “Vascular development and disorders:Molecular analysis and pathogenic insights,” Kidney International,53:1519-1549 (1998) at pages 1521-1522. Thus, while all VEGF-A isoformsare active and within the scope of angiogenic agents of the presentinvention, the highly basic and heparin binding carboxy terminus ofVEGF-A is important to maximizing activity. Although the mechanism bywhich VEGF-A stimulates angiogenesis is not known, Banai suggests thatVEGF-A promotes angiogenesis in part via stimulation of endothelialrelease of PDGF. Banai, et al., “Angiogenic-Induced Enhancement ofCollateral Blood Flow to Ischemic Myocardium by Vascular EndothelialGrowth Factor in Dogs,” Circulation, 89(5):2183-2189 (May 1994). VEGF-Abinds to the VEGF receptor-1 (VEGFR-1 or FLT1) and to the VEGFreceptor-2 (VEGFR-2 or FLK1).

Human VEGF-B, which is found in abundance in heart and skeletal muscle,is a known nonglycosylated highly basic heparin binding protein that hasthe amino acid sequence shown in FIG. 1 of Olofsson, et al., “Vascularendothelial growth factor B, a novel growth factor for endothelialcells,” PNAS USA 93:2576-2581 (1996). Like the VEGF-As, VEGF-B isexpressed as a prohormone and has 188 amino acid residues of whichresidues 1-21 are a putative leader sequence and thus are not necessaryfor angiogenic activity. Id. Hence, mature human VEGF-B comprises the167 residues that follow the putative leader sequence. Olofsson atFIG. 1. The human prohormone VEGF-B also has 88% sequence identity tomurine prohormone VEGF-B, varying at residue positions 12, 19, 20, 26,28, 30, 33, 37, 43, 57, 58, 63, 65, 105, 130, 140, 144, 148, 149, 165,168, 186 and 188 in a conserved manner. Olofsson at page 2577, col. 2,and FIGS. 1 and 2 therein. The differences in residues in going frommature human VEGF-B to mature murine VEGF-B are as follows: 5 Pro→Phe, 7Ala→Gly, 9 Gly→Ser, 12 Arg→Lys, 16 Ser→Pro, 22 Thr→Ala, 36 Thr→Ser, 37Val→Met, 42 Thr→Asn, 44 Ala→Val, 86 Arg→Gln, 119 Asp→Glu, 129 Pro→Ile,133 Arg→Pro, 137 His→Arg, 138 His→Arg, 165 Ser→Arg, 168 Arg→His, 165Leu→Pro, and 167 Arg→Lys. Accordingly, an angiogenic agent of thepresent invention includes a human VEGF-B mutein having a conservativesubstitution at one or more of the above-referenced residue positions.Preferably, the conservative substitution is one or more of the abovereferenced differences in the second preceding sentence above.

VEGF-C, which is expressed most prominantly in the heart, lymph nodes,placenta, ovary, small intestine and thyroid, is induced by a variety ofgrowth factors, inflammatory cytokines and hypoxia. VEGF-C isrecombinantly expressed as disclosed in Joukov et al. and has the aminoacid sequence disclosed at page 291 and FIG. 3 therein. See Joukov etal., “A novel vascular endothelial growth factor, VEGF-C, is a ligandfor the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases,” TheEMBO Journal, 15(2):290-298 (1996); also Ferrara, et al., “The Biologyof Vascular Endothelial Growth Factor,” Endocrine Reviews, 18(1):4-25(1997) at FIG. 3. VEGF-C is the largest member of the VEGF family,having 399 amino acid residues and 32% homology to VEGF-A. See Ferrara(1997) at page 11, col. 1. The carboxy end of VEGF-C contains 180residues of insert (at residue positions 213-295) that are not found inthe other VEGFs. See Joukov et al. (1996) at FIG. 3: or Ferrara, et al.,“The Biology of Vascular Endothelial Growth Factor,” Endocrine Reviews,18(1):4-25 (1997) at FIG. 4. Given its large size, VEGF-C would be theleast desirable member of the VEGF family. However, deletion muteins ofVEGF-C, lacking residues 213-295, or fragments thereof, lacking one ormore residues at the N-terminus, up to residues 1-28 are also within thescope of the term angiogenic factor as used in the present invention.VEGF-C binds to VEGFR-2 (previously known as flt-1 and KDR/Flk-1) and toVEGFR-3 (also known as Flt4). See Joukov et al. (1996).

VEGF-D, which is the most recent member of the VEGF family to bediscovered, is encoded by the cDNA and has the amino acid sequence shownin FIG. 2 of commonly assigned U.S. Ser. No. 09/043,476, filed Mar. 18,1998; and corresponding WO 97/12972 which was published on Apr. 10,1997. VEGF-D is a dimerizing protein having 304 amino acid residues. Thecore of VEGF-D is highly conserved relative to the other VEGF proteins.More importantly, it contains the 15 cysteine residues at residuepositions 111, 136, 142, 145, 146, 153, 189, 191, 258, 269, 271, 273,300, 312 and 314 that are highly conserved throughout the VEGFs andPDGFs. Overlapping comparisons of the amino acid sequences of the VEGFsand some of the PDFs, showing the conserved areas, are found in Ferrara,et al., “The Biology of Vascular Endothelial Growth Factor,” EndocrineReviews, 18(1):4-25 (1997) at FIG. 4; in WO 97/12972 and its U.S.equivalent U.S. Ser. No. 09/043,476 at FIG. 3; and WO 98/02543 at FIG.3. Biologically active alleles and fragments of the VEGF-D are known inthe art. In one example, WO 98/07832 discloses a biologically activehuman VEGF-D that was isolated from lung that differs from the VEGF-D ofWO 97/12972 by having the following variations at the designated residuepositions: 56 Thr→Ile, 151 Phe→Leu, 152 Met→Ile, 261 Asp→His, 264 GluePhe, and 297 Glu→Leu. Accordingly, it is within the scope of the presentinvention that the angiogenic agent include muteins of VEGF-D thatinclude one or more of the above-referenced amino acid substitutions ora conservative substitution at one or more of the above-referencedresidue sites. Such muteins are made by site directed mutagenesis, whichis a standard technique in the art. In addition, a biologically activeVEGF-D that was isolated from human breast tissue lacked the first 30amino acids. See WO 98/24811. Accordingly, it is within the scope ofthis invention that angiogenic agent include fragments of VEGF-D thatlack amino acid residues 1-30 of the mature VEGF-D. Moreover, insofar asresidues 109-315 of mature VEGF-D contain the highly conserved regionthat is responsible for dimerization and binding to receptor, it is alsowithin the scope of the present invention that the angiogenic agentinclude an N-truncated and/or C-truncated VEGF-D comprising residues109-315 of the mature hormone of FIG. 2 of WO 97/12972 or correspondingU.S. Ser. No. 09/043,476.

TGF-β1 is a member of the TGF-β superfamily, having two dozen members.The various members of the TGF-β superfamily are homo- or hetero-dimersof a mature protein having 110-140 amino acid residues and at leastseven cysteines. Six of the cysteines form internal disulfides and theseventh forms a disulfide bond that links the two monomers together. SeeKingsley, D. M., “The TGF-β superfamily: new members, new receptors, andnew genetic tests of function in different organisms,” Genes andDevelop., 8:133-146 (1994). The TGF-β1 monomer, like the other monomersof the TGF-β superfamily, has a structural similarity with PDGF, albeitless than 10%. The monomer for human TGF-β1 is a known 112 residueprotein encoded by the cDNA and having the deduced amino acid sequenceshown in FIG. 1B(III) of U.S. Pat. No. 4,886,747, entitled “Nucleic AcidEncoding TGF-β and Its Uses,” which issued on Dec. 12, 1989 to Deryncket al. and discloses methods for expressing recombinant TGF-β1. AlthoughTGF-β1 has 112 amino acid residues, only the sequence of residuescorresponding to residue positions 16-31 (i.e., CVRQLYIDFRKDLGWK) of themature TGF-β1 (see e.g., FIG. 1B(III) of the '747 patent) is necessaryfor activity. See U.S. Pat. No. 5,658,883, entitled “Biologically ActiveTGF-β1 Peptides,” which issued on Aug. 17, 1997 to Ogawa et al. A largerdimerized fragment of mature human TGF-β1, corresponding to residues16-47 (i.e., CVRQLYIDFRKDLGWKWIHEPKGYHANFCLGP), exhibited similaractivity to dimers of the 16-31 fragment and to dimers of mature TGF-β1.The 16-31 residue fragment is dimerized by forming disulfide bondsbetween the amino terminal cysteines of two monomeric subunits. The16-47 residue fragment is dimerized by forming disulfide bonds betweenthe amino terminal cysteines, the carboxy terminal cysteines or both oftwo monomer subunits. Thus, it is within the scope of the presentinvention that a fragment of TGF-β1 need only comprise residues 16-31 ofthe mature human TGF-β1 to be an active fragment. In addition todirectly inducing angiogenesis, there is speculation that TGF-β1 mayinduce angiogenesis indirectly in vivo by affecting inflammatory orconnective tissue cells, which in turn can produce angiogenic molecules,such as VEGF-A, PDGF, FGF-2, etc. See Carmeliet (1998).

Another angiogenic agent suitable for use in the compositions and methodof the present invention is FGF. By the term “FGF,” as used herein, ismeant a fibroblast growth factor protein that also has angiogenicactivity (such as FGF-1, FGF-2, FGF-4, FGF-6, FGF-8, FGF-9 or FGF-98) oran angiogenically active fragment or mutein thereof. Typically, the FGFis human (h) FGF-1, bovine (b) FGF-1, hFGF-2, bFGF-2, hFGF-4 or hFGF-5.In an alternative embodiment, the active agent in the unit dose ishFGF-6, mFGF-8, hFGF-9 or hFGF-98.

The amino acid sequences and methods for making many of the FGFs thatare employed in the unit dose pharmaceutical composition and method ofthe present invention are well known in the art. In particular,references disclosing the amino acid sequence and recombinant expressionof FGF 1-9 and FGF-98 are discussed sequentially below.

FGF-1: The amino acid sequence of hFGF-1 and a method for itsrecombinant expression are disclosed in U.S. Pat. No. 5,604,293(Fiddes), entitled “Recombinant Human Basic Fibroblast Growth Factor,”which issued on Feb. 18, 1997. See FIG. 2 d of the '293 patent. Thisreference and all other references herein, whether cited before or afterthis sentence, are expressly incorporated herein by reference in theirentirety. The amino acid sequence of bFGF-1 is disclosed in U.S. Pat.No. 5,604,293 (Fiddes) at FIG. 1 b, as is a method for its expression.The mature forms of both hFGF-1 and bFGF-1 have 140 amino acid residues.bFGF-1 differs from hFGF-1 at 19 residue positions: 5 Pro→Leu, 21His→Tyr, 31 Tyr→Val, 35 Arg→Lys, 40 Gln→Gly, 45 Gln→Phe, 47 Ser→Cys, 51Tyr→Ile, 54 Tyr→Val, 64 Tyr→Phe, 80 Asn→Asp, 106 Asn→His, 109 Tyr→Val,116 Ser→Arg, 117 Cys→Ser, 119 Arg→Leu, 120 Gly→Glu, 125 Tyr→Phe and 137Tyr→Val. In most instances, the differences are conserved. Further, thedifferences at residue positions 116 and 119 merely interchange theposition of the Arg.

FGF-2: The cDNA sequence (SEQ ID NO: 4) encoding the full length 155residue human FGF-2 (SEQ ID NO: 5) and methods for recombinantexpression of human FGF-2 (hFGF-2) are disclosed in U.S. Pat. No.5,439,818 (Fiddes) entitled “DNA Encoding Human Recombinant BasicFibroblast Growth Factor,” which issued on Aug. 8, 1995 (see FIG. 4therein), and in U.S. Pat. No. 5,514,566 (Fiddes), entitled “Methods ofProducing Recombinant Fibroblast Growth Factors,” which issued on May 7,1996 (see FIG. 4 therein). Human FGF-2 also has an active N-truncated146 residue form of SEQ ID NO: 6, that lacks the first nine residuesfrom the N-terminus of SEQ ID NO: 5. This truncated form is readilyproduced by making appropriate deletions to the 5′ end of the cDNA ofSEQ ID NO: 4, using art-known techniques. The cDNA sequence (SEQ IDNO: 1) encoding bovine FGF-2 (SEQ ID NO: 2) and various methods for itsrecombinant expression are disclosed in U.S. Pat. No. 5,155,214,entitled “Basic Fibroblast Growth Factor,” which issued on Oct. 13,1992. When the 146 residue forms of hFGF-2 and bFGF-2 are compared,their amino acid sequences are nearly identical with only two residuesthat differ. In particular, in going from hFGF-2 to bFGF-2, the soledifferences occur at residue positions 112(Thr→Ser) and 128(Ser→Pro).

FGF-3: FGF-3 was first identified as an expression product of a mouseint-2 mammary tumor and its amino acid sequence is disclosed in Dicksonet al., “Potential Oncogene Product Related to Growth Factors,” Nature326:833 (Apr. 30, 1987). FGF-3, which has 243 residues when theN-terminal Met is excluded, is substantially longer than both FGF-1(human and bovine) and FGF-2 (human and bovine). A comparison of aminoacid residues for mFGF-3 relative to bFGF-1 and bFGF-2 is presented inoverlap fashion in Dickson, et al. (1987). When the amino acid sequenceof mFGF-3 is compared to bFGF-1 and bFGF-2, FGF-3 has 5 locationscontaining residue inserts relative to both FGF-1 and FGF-2. The mostsignificant of these inserts is a 12 and a 14 residue insert relative toFGF-2 and FGF-1, respectively, beginning at residue position 135 ofFGF-3. Allowing for the inserts, Dickson discloses that mFGF-3 has 53residue identities relative to FGF-1 and 69 residue identities relativeto FGF-2. In addition, FGF-3 contains a hydrophobic N-terminal extensionof 10 residues relative to the N-terminus of the signal sequence in bothFGF-1 and FGF-2. Relative to the C-terminus of bFGF-1 and bFGF-2, mFGF-3contains an approximately 60 residue extension. It is unlikely that theC-terminal extension of mFGF-3 is necessary for activity. More likely,it is a moderator of activity by conferring receptor specificity on theFGF.

FGF-4: The amino acid sequence for the hst protein, now known as hFGF-4,was first disclosed by Yoshida, et al., “Genomic Sequence of hst, aTransforming Gene Encoding a Protein Homologous to Fibroblast GrowthFactors and the int-2-Encoded Protein,” PNAS USA, 84:7305-7309 (October1987) at FIG. 3. Including its leader sequence, hFGF-4 has 206 aminoacid residues. When the amino acid sequences of hFGF-4, hFGF-1, hFGF-2and mFGF-3 are compared, residues 72-204 of hFGF-4 have 43% homology tohFGF-2; residues 79-204 have 38% homology to hFGF-1; and residues 72-174have 40% homology to mFGF-3. A comparison of these four sequences inoverlap form is shown in Yoshida (1987) at FIG. 3. Further, the Cys atresidue positions 88 and 155 of hFGF-4 are highly conserved amonghFGF-1, hFGF-2, mFGF-3 and hFGF-4 and are found in a homologous region.

The two putative cell-binding sites of hFGF-2 occur at residue positions36-39 and 77-81 thereof. See Yoshida (1987) at FIG. 3. The two putativeheparin-binding sites of hFGF-2 occur at residue positions 18-22 and107-111 thereof. See Yoshida (1987) at FIG. 3. Given the substantialsimilarity between the amino acid sequences for human and bovine FGF-2,we would expect the cell-binding sites for bFGF-2 to also be at residuepositions 36-39 and 77-81 thereof, and the heparin-binding sites to beat residue positions 18-22 and 107-111 thereof. In relation to hFGF-1,the putative cell-binding sites occur at residues 27-30 and 69-72, andthe putative heparin-binding sites occur at residues 9-13 and 98-102.Insofar as mature bFGF-1 has the identical amino acids at residuepositions 9-13, 27-30, 69-72 and 98-102 as does mature hFGF-2, bFGF-1would be expected to have the same cell- and heparin-binding sites asdoes hFGF-1.

FGF-5: The cDNA and deduced amino acid sequence for hFGF-5 are disclosedin Zhan, et al., “The Human FGF-5 Oncogene Encodes a Novel ProteinRelated to Fibroblast Growth Factors,” Molec. and Cell. Biol.,8(8):3487-3495 (August 1988) at FIG. 1. Zhan also discloses a method forcloning hFGF-5. The Applicants also sequenced hFGF-5 and obtained anamino acid sequence which differed from Zhan's sequence at residueposition 236 (having a Lys instead of the Zhan's Asn) and at residueposition 243 (having a Pro instead of Zhan's Ser). Both amino acidsequences for hFGF-5 have 266 amino acid residues that include a leadersequence of 67 residues upstream of the first residue of mature FGF-2and a tail sequence that extends about 47 residues beyond the C-terminusof hFGF-2. A comparison between the amino acid sequences of hFGF-1,hFGF-2, mFGF-3, hFGF-4 and FGF-5 is presented in FIG. 2 of Zhan (1988).In FIG. 2 of Zhan, hFGF-1, hFGF-2, mFGF-3 and hFGF-4 are identified asaFGF (i.e., acidic FGF), bFGF (i.e., basic FGF), int-2, and hstKS3,respectively, i.e., by their original names. In the above referencedcomparison, two blocks of FGF-5 amino acid residues (90 to 180 and187-207) showed substantial homology to FGF 1-4, i.e., 50.4% with FGF-4,47.5% with FGF-3, 43.4% with FGF-2 and 40.2% with hFGF-1. See Zhan(1988) at FIG. 2. U.S. Pat. No. 5,155,217 (Goldfarb) and U.S. Pat. No.5,238,916 (Goldfarb), which correspond to the Zhan publication, refer tothe FGF-5 of Zhan as FGF-3. However, the art (as evidenced by Coulierbelow) has come to recognize that the hFGF of Zhan (and of the Goldfarbpatents) as FGF-5 and not as FGF-3. The two Goldfarb patents contain thesame amino acid sequence for hFGF-5 as reported above by Zhan.

FGF-6: The cDNA and deduced amino acid sequence for hFGF-6 are disclosedin Coulier et al., “Putative Structure of the FGF-6 Gene Product andRole of the Signal Peptide,” Oncogene 6:1437-1444 (1991) at FIG. 2.Coulier also discloses a method for cloning FGF-6. hFGF-6 is one of thelargest of the FGFs, having 208 amino acid residues. When the amino acidsequences of human FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6 and FGF-7are compared, there are strong similarities in the C-terminal two-thirdsof the molecules (corresponding e.g., to residues 78-208 of hFGF-6. Inparticular, 23 residues of FGF-6, including the two cysteines at residuepositions 90-157 of hFGF-6 were identical between the seven members ofthe family. This number increases to 33 residues when conserved aminoacid residues are considered. The overall similarities between theseseven human FGFs ranged from 32% to 70% identical residues and 48% to79% conserved residues for the C-terminal two-thirds of the molecules.The sequence comparisons of hFGF-1 to hFGF-5 and hFGF-7, relative tohFGF-6, are shown in Table 1 herein. TABLE 1 Amino Acid SequenceComparison of hFGF-6 With Other hFGFs Identical Conserved IdenticalConserved Residues* Residues** Residues* (%) Residues** (%) hFGF-4 91103  70 79 hFGF-5 64 82 49 63 hFGF-3 55 78 42 60 hFGF-2 54 69 42 53hFGF-7 47 68 36 52 hFGF-1 42 62 32 48*Number and percentages of identical or conserved residues werecalculated for the C-terminal two-thirds of the hFGF6 molecule (residues78-208).**Conserved residues are defined according to the structure-geneticmatrix of Feng et al., J. Mol. Evol., 21: 112-125 (1985).

Referring to Table 1, FGF-6 has the highest correspondence (91 identicalresidues/103 conserved residues) with FGF-4. This amounts to 70%identical and 79% conserved residues. hFGF-6 differed most from hFGF-3,hFGF-2, hFGF-7 and hFGF-1, with 42, 42, 36 and 32; identical residues,respectively.

An overlaid comparison of the amino acid sequences of FGFs 1-7 is shownin FIG. 3 of incorporated Coulier (1991). FIG. 3 of Coulier shows thatwhen in the C-terminal two thirds of the FGF molecules are aligned,there are 23 residue positions wherein the residues from all seven FGFmembers are identical. There are also ten residue positions whereinresidues from all seven FGF members are conserved. Coulier (1991) atFIG. 3. In combination, these identical and conserved residues formabout 6 locations of three to five residues on the terminal two thirdsof each of the FGFs 1-7, wherein three to five residues are groupedtogether in all seven species of human FGF (i.e., hFGF 1-7).

FGF-7: The amino acid sequence of hFGF-7 is well-known in the art anddisclosed in Miyamoto, et al., “Molecular Cloning of a Novel CytokinecDNA Encoding the Ninth Member of the Fibroblast Growth Factor Family,Which Has a Unique Secretion Property,” Mol. and Cell. Biol.13(7):4251-4259 (1993) at FIG. 2. In Miyamoto, the hFGF-7 was referredto by its older name “KGF”. FGF-7 has 191 amino acid residues. Acomparison of the amino acid sequence of hFGF-7 to the amino acidsequences of hFGF 1-6 and hFGF-9 shows that the carboxy terminal twothirds of the FGF-7 has comparable homology with the distal two thirdsof the other members of the group. See Miyamoto (1993) at page 4254(FIG. 2).

FGF-8: The cDNA and deduced amino acid sequence of mFGF-8 is well-knownin the art and disclosed in Tanaka et al., “Cloning and Characterizationof an Androgen-induced Growth Factor Essential for the Growth of MouseMammary Carcinoma Cells,” PNAS USA, 89:8928-8932 (1992) at FIG. 2.Tanaka also discloses a method for making recombinant FGF-8. The mFGF-8of Tanaka has 215 amino acid residues. MacArthur, et al., “FGF-8isoforms activate receptor splice forms that are expressed inmesenchymal regions of mouse development,” Development, 121:3603-3613(1995) discloses that FGF-8 has 8 different isoforms that differ at themature N-terminus but that are identical over the C-terminal region. The8 isoforms arise because FGF-8 has 6 exons of which the first four(which correspond to the first exon of most other FGF genes) result inalternative splicing.

FGF-9: The cDNA and deduced amino acid sequences of human and murineFGF-9 are known in the art and methods for their recombinant expressionare disclosed in Santos-Ocampo, et al., “Expression and BiologicalActivity of Mouse Fibroblast Growth Factor,” J. Biol. Chem.,271(3):1726-1731 (1996). Both the human and murine FGF-9 molecules have208 amino acid residues and sequences that differ by only two residues.In particular, hFGF-9 has Asn and Ser at residues 9 and 34,respectively, whereas mFGF-9 has Ser and Asn, respectively. FGF-9 hascomplete preservation of the conserved amino acids that define the FGFfamily. Santos-Ocampo (1996) at page 1726. Half-maximal activation ofFGF-9 is seen at 185 ng/ml heparin, whereas half-maximal activation ofFGF-1 is seen at 670 ng/ml heparin. Santos-Ocampo (1996) at page 1730.When compared to FGF-1, both FGF-2 and FGF-9 require lower heparinconcentrations for optimal activity.

FGF-98: The cDNA and amino acid sequence of hFGF-98 and a method for itsrecombinant expression are disclosed in provisional patent applicationSer. No. 60/083,553 which is hereby incorporated herein by reference inits entirety. hFGF-98, which is also known as hFGF-18, has 207 aminoacid residues. Thus, hFGF-6 (207 residues), hFGF-9 (208 residues) andhFGF-98 (207 residues) are similar in size.

FGFs differentially bind to and activate one or more of four relatedtransmembrane receptors which in turn mediate a biological response. TheFGF receptors (“FGFR”) are members of the tyrosine kinase receptorsuperfamily. The extracellular domain of the FGFR comprises 2-3immunoglobulin-like (“Ig-like”) domains that are differentiallyexpressed as a result of alternative splicing. Another alternativesplicing event can also alter the sequence of the carboxy-terminal halfof the Ig-like domain m without altering the reading frame.Santos-Ocampo (1996). The two splice forms, which are referred to as “b”and “c”, occur for FGFRs 1, 2, 3 but not 4. A more detailed descriptionof the FGFR is found in Mathieu, et al, “Receptor Binding and MitogenicProperties of Mouse Fibroblast Growth Factor 3,” J. Biol. Chem.,270(41):24197-24203 (1995). The ability of FGF 1-9 to differentiallystimulate FGFRs was receptor dependent as reported by Ornitz et al., J.Biol. Chem., 271(25):15292-15297 (1996). In Ornitz, the cell line BaF3was divided into fractions and each fraction was transfected to expressone of the following FGF receptors: FGFR1b, FGFR1c, FGFR2b, FGFR2c,FGFR3b, FGFR3c and FGF4 (minus one Ig-like domain). Thereafter, thetransformed cell lines were exposed to one of FGF 1-9 (5 nM) and heparin(2 μg/ml) as a cofactor. The mitogenic response was then measured byincorporation of [³H] thymidine. The results in cpm are as follows:

-   -   1. FGFR1b: similar mitogenic responses were produced by HFGF-1        (32,000 cpm) and hFGF-2 (28,000 cpm) with the next highest        responses by mFGF-3 (about 16,000 cpm) and hFGF-4 (15,000 cpm);    -   2. FGFR1c: similar mitogenic responses were produced by hFGF-1,        hFGF-2, hFGF-4, hFGF-5, and hFGF-6 (about 36,000 cpm), with        mFGF-9 producing the only other significant response (about        19,000 cpm);    -   3. FGFR2b: best mitogenic responses were by hFGF-7 (14,000 cpm),        hFGF-1 (12,500 cpm) and mFGF-3 (9,500 cpm);    -   4. FGFR2c: best mitogenic responses were by hFGF-4 (21,000 cpm),        mFGF-9 (20,000 cpm), hFGF-6 (16,500 cpm), hFGF-1 (16,000 cpm),        hFGF-2 (14,500 cpm), hFGF-5 (9,500 cpm), and mFGF-8 (9,000 cpm);    -   5. FGFR3b: mitogenic responses only by hFGF-1 (37,000 cpm) and        mFGF-9 (26,000 cpm);    -   6. FGFR3c: best mitogenic responses by hFGF-1 (39,000 cpm),        hFGF-2 (34,000 cpm), hFGF-4 (33,000 cpm), mFGF-8 (32,500 cpm),        mFGF-9 (31,000 cpm), hFGF-5 (16,000 cpm) and hFGF-6 (13,000        cpm);    -   7. FGFR4): best mitogenic responses by hFGF-2 (29,000 cpm),        hFGF-4 and hFGF-6 (27,000 cpm), mFGF-8 (25,000 cpm), mFGF-1        (24,000 cpm), and hFGF-9 (20,000 cpm) with all others being        6,000 cpm or less.

As reflected above, only FGF-1 induces a significant mitogenic responsein all of the receptors tested. Thus, FGF-1 can be thought of as auniversal ligand with N- and C-terminal additions to the molecule givingrise to receptor specificity associated with the other FGF. Given thepotential for diverse responses in vivo by systemically administeredFGF, the present invention minimizes the potential for systemicresponses by localized administration, and by discovering theappropriate dosage for the localized administration, i.e., byadministering a therapeutically effective amount of a FGF into at leastone coronary artery of a patient in need of treatment for CAD. In theExamples that follow, bFGF-2 was administered in vivo to rats, pigs andhumans, and tested for angiogenic activity. The bFGF-2 of the Exampleswas made as described in U.S. Pat. No. 5,155,214 (“the '214 patent”). Inthe method of the '214 patent, a cDNA encoding bFGF (hereinafter“FGF-2”) is inserted into a cloning vector, such as pBR322, pMB9, ColE1, pCRI, RP4 or λ-phage, and the cloning vector is used to transformeither a eukaryotic or prokaryotic cell, wherein the transformed cellexpresses the FGF-2. In one embodiment, the host cell is a yeast cell,such as Saccharomyces cerevisiae. The resulting full length FGF-2 thatis expressed has 146 amino acids in accordance with sequence shown atcol. 6 of the '214 patent. Although the resulting FGF-2 has fourcysteines, i.e., at residue positions 25, 69, 87 and 92, there are nointernal disulfide linkages. [The '214 patent at col. 6, lines 59-61.]However, in the event that cross-linking occurred under oxidativeconditions, it would likely occur between the two Cys residues atpositions 25 and 69, respectively.

Bovine FGF-2 (bFGF-2), like the corresponding human FGF-2 (hFGF-2), isinitially synthesized in vivo as a polypeptide having 155 amino acidresidues. Abraham et al. “Human Basic Fibroblast Growth Factor:Nucleotide Sequence and Genomic Organization,” EMBO J., 5(10):2523-2528(1986). When the 146 residue bFGF-2 (SEQ ID NO: 2) of the examples iscompared to the full length 155 residue bFGF-2 of Abraham, Applicants'bFGF-2 (SEQ ID NO: 2) lacks the first nine amino acid residues, i.e.,Met-Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu (SEQ ID NO: 3) found at theN-terminus of Abraham's full length molecule. As discussed above, maturebFGF-2 differs from mature hFGF-2 in only two residue positions. Inparticular, the amino acids at residue positions 112 and 128 of themature bFGF-2 (SEQ ID NO: 2), are Ser and Pro, respectively, whereas incorresponding mature hFGF-2 (SEQ ID NO: 6), they are Thr and Ser,respectively. In view of this substantial structural identity (i.e.,greater than 97% identity) between bFGF and hFGF-2, the in vivo clinicalresults that are provided in the Examples, and discussed elsewhereherein on the angiogenic activity, dosage and mode of administeringrecombinant bFGF-2 should be directly applicable to recombinant hFGF-2(collectively “FGF-2”).

The recombinant bFGF-2 (SEQ ID NO: 2) of the Examples was purified topharmaceutical quality (98% or greater purity) using the techniquesdescribed in detail in U.S. Pat. No. 4,956,455 (the '455 patent),entitled “Bovine Fibroblast Growth Factor” which issued on Sep. 11, 1990and which was incorporated herein by reference in its entirety. Inparticular, the first two steps employed in the purification of therecombinant bFGF-2 of Applicants' unit dose are “conventionalion-exchange and reverse phase HPLC purification steps as describedpreviously.” [The '455 patent, citing to Bolen et al., PNAS USA81:5364-5368 (1984).] The third step, which the '455 patent refers to asthe “key purification step” [see the '455 patent at col. 7, lines 5-6],is heparin-SEPHAROSE® affinity chromatography, wherein the strongheparin binding affinity of the FGF-2 is utilized to achieve severalthousand-fold purification when eluting at approximately 1.4M and 1.95MNaCl [the '455 patent at col. 9, lines 20-25]. Polypeptide homogeneitywas confirmed by reverse-phase high pressure liquid chromatography(RP-HPLC). Buffer exchange was achieved by SEPHADEX® G-25(M) gelfiltration chromatography.

In addition to the above-described FGFs, the angiogenic agent of thecompositions and the method of the present invention also comprises an“angiogenically active fragment” of any one of the above-described FGFs.In its simplest form, the angiogenic fragment is made by the removal ofthe N-terminal methionine, using well-known techniques for N-terminalMet removal, such as treatment with a methionine aminopeptidase. Asecond desirable truncation comprises an FGF without its leadersequence. Those skilled in the art recognize the leader sequence as theseries of hydrophobic residues at the N-terminus of a protein thatfacilitate its passage through a cell membrane but that are notnecessary for activity and that are not found on the mature protein.

Preferred truncations on the FGFs are determined relative to maturehFGF-2 (SEQ ID NO: 6) or the analogous bFGF-2 (SEQ ID NO: 2) having 146residues. As a general rule, the amino acid sequence of an FGF isaligned with FGF-2 to obtain maximum homology. Portions of the FGF thatextend beyond the corresponding N-terminus of the aligned FGF-2 aregenerally suitable for deletion without adverse effect. Likewise,portions of the FGF that extend beyond the C-terminus of the alignedFGF-2 are also capable of being deleted without adverse effect.

Fragments of FGF that are smaller than those described above are alsowithin the scope of the present invention so long as they retain thecell-binding portions of FGF and at least one heparin-binding segment.In the case of mature FGF-2 having residues 1-146, the two putativecell-binding sites occur at residue positions 36-39 and 77-81 thereof.See Yoshida, et al., “Genomic Sequence of hst, a Transforming GeneEncoding a Protein Homologous to Fibroblast Growth Factors and theint-2-Encoded Protein,” PNAS USA, 84:7305-7309 (October 1987) at FIG. 3.The two putative heparin-binding sites of hFGF-2 occur at residuepositions 18-22 and 107-111 thereof. See Yoshida (1987) at FIG. 3. Giventhe substantial sequence identity between the amino acid sequences forhFGF-2 and bFGF-2, we expect that the cell-binding sites for bFGF-2 arealso at residue positions 36-39 and 77-81 thereof, and that theheparin-binding sites are at residue positions 18-22 and 107-111thereof. Consistent with the above, it is well known in the art thatN-terminal truncations of bFGF-2 do not eliminate its angiogenicactivity in cows. In particular, the art discloses several naturallyoccurring and biologically active fragments of bFGF-2 that haveN-terminal truncations relative to the 146-residue mature FGF-2. Anactive and N-truncated FGF-2 fragment having residues 12-146 of matureFGF-2 was found in bovine liver and another active and N-truncated FGF-2fragment, having residues 16-146 of mature FGF-2 was found in the bovinekidney, adrenal glands and testes. [See U.S. Pat. No. 5,155,214 at col.6, lines 41-46, citing to Ueno, et al., Biochem and Biophys Res. Comm.,138:580-588 (1986).] Likewise, other fragments of FGF-2 that are knownto have FGF activity are FGF-2 (24-120)-OH and FGF-2 (30-110)-NH₂. [U.S.Pat. No. 5,155,214 at col. 6, lines 48-52.] These latter fragmentsretain both of the cell binding portions of FGF-2 (residues 36-39 and77-81) and one of the heparin binding segments (residues 107-111).Accordingly, the angiogenically active fragments of an FGF typicallyencompass those terminally truncated fragments of an FGF that whenaligned to mature FGF-2 (having residues 1-146) to maximize homology,have at least residues that correspond to residue positions 30-110 ofFGF-2; more typically, at least residues that correspond to residues18-146 of FGF-2.

In addition to the above described FGFs, the angiogenic agent of theunit dose, compositions and method of the present invention alsocomprises an “angiogenically active . . . mutein” thereof. By the term“angiogenically active . . . mutein,” as used in conjunction with anFGF, is meant a mutated form of the naturally occurring FGF that retainsat least 65% sequence identity (preferably 75%, more preferably 85%,most preferably 90% sequence identity) and at least 80% of theangiogenic activity of the respective FGF, wherein sequence identity isdetermined by the Smith-Waterman homology search algorithm (Meth. Mol.Biol. 70:173-187 (1997)) as implemented in MSPRCH program (OxfordMolecular) using an affine gap search with the following searchparameters: gap open penalty of 12, and gap extension penalty of 1.Preferably, the mutations are “conservative amino acid substitutions”using L-amino acids, wherein one amino acid is replaced by anotherbiologically similar amino acid. As previously noted, conservative aminoacid substitutions are those that preserve the general charge,hydrophobicity/hydrophilicity, and/or steric bulk of the amino acidbeing substituted. Examples of conservative substitutions are thosebetween the following groups: Gly/Ala, Val/Ile/Leu, Lys/Arg, Asn/Gln,Glu/Asp, Ser/Cys/Thr, and Phe/Trp/Tyr. In the case of FGF-2, an exampleof such a conservative amino acid substitution includes the substitutionof serine for one or both of the cysteines at residue positions whichare not involved in disulfide formation, such as residues 87 and 92 inmature FGF-2 (having residues 1-146). Preferably, substitutions areintroduced at the N-terminus, which is not associated with angiogenicactivity. However, as discussed above, conservative substitutions aresuitable for introduction throughout the molecule.

One skilled in the art, using art known techniques, is able to make oneor more point mutations in the DNA encoding any of the FGFs to obtainexpression of an FGF polypeptide mutein (or fragment mutein) havingangiogenic activity for use within the unit dose, compositions andmethod of the present invention. To prepare an angiogenically activemutein of an FGF, one uses standard techniques for site directedmutagenesis, as known in the art and/or as taught in Gilman, et al.,Gene, 8:81 (1979) or Roberts, et al., Nature, 328:731 (1987), tointroduce one or more point mutations into the cDNA that encodes theFGF.

Thus, the pharmaceutical composition of the present invention comprisesan angiogenically effective amount of an angiogenic agent, in apharmaceutically acceptable carrier, the angiogenically effective amountbeing in the range from about 5 ng to less than about 135,000 ng, theangiogenic agent being platelet derived growth factor (PDGF), vascularendothelial growth factor-A (VEGF-A), VEGF-D, fibroblast growth factor(FGF), or an angiogenically active fragment or mutein thereof. In apreferred embodiment, the angiogenic agent of the pharmaceuticalcomposition is human VEGF-A, human VEGF-D, FGF or an angiogenicallyactive fragment or mutein thereof. More preferably, the angiogenic agentof the pharmaceutical composition is an FGF, such as FGF-1, FGF-2 orFGF-5, or an angiogenically active fragment or mutein thereof. Mostpreferably, the angiogenic agent of the pharmaceutical composition is anFGF-2, or an angiogenically active fragment or mutein thereof.

The unit dose pharmaceutical composition of the present inventioncontains as its second recited component a “pharmaceutically acceptablecarrier.” By the term “pharmaceutically acceptable carrier” as usedherein is meant any of the carriers or diluents that are well known inthe art for the stabilization and/or administration of a proteinaceousmedicament that does not itself induce the production of antibodiesharmful to the patient receiving the composition, and which may beadministered without undue toxicity. The choice of the pharmaceuticallyacceptable carrier and its subsequent processing enables the unit dosecomposition of the present invention to be provided to the treatingphysician in either liquid or solid form. However, the unit dosecomposition of the present invention is converted to liquid form beforeit is administered to the patient by injection into the myocardium.

When the unit dose pharmaceutical composition is in liquid form, thepharmaceutically acceptable carrier comprises a stable carrier ordiluent suitable for intravenous (“IV”) or intracoronary (“IC”)injection or infusion. Suitable carriers or diluents for injectable orinfusible solutions are nontoxic to a human recipient at the dosages andconcentrations employed, and include sterile water, sugar solutions,saline solutions, protein solutions or combinations thereof.

Typically, the pharmaceutically acceptable carrier includes a buffer andone or more stabilizers, reducing agents, anti-oxidants and/oranti-oxidant chelating agents. The use of buffers, stabilizers, reducingagents, anti-oxidants and chelating agents in the preparation of proteinbased compositions, particularly pharmaceutical compositions, iswell-known in the art. See, Wang et al., “Review of Excipients and pHsfor Parenteral Products Used in the United States,” J. Parent. DrugAssn., 34(6):452-462 (1980); Wang et al., “Parenteral Formulations ofProteins and Peptides: Stability and Stabilizers,” J. Parent. Sci. andTech., 42:S4-S26 (Supplement 1988); Lachman, et al., “Antioxidants andChelating Agents as Stabilizers in Liquid Dosage Forms-Part I,” Drug andCosmetic Industry, 102(1): 36-38, 40 and 146-148 (1968); Akers, M. J.,“Antioxidants in Pharmaceutical Products,” J. Parent. Sci. and Tech.,36(5):222-228 (1988); and Methods in Enzymology, Vol. XXV, Colowick andKaplan Eds., “Reduction of Disulfide Bonds in Proteins withDithiothreitol,” by Konigsberg, pages 185-188. Suitable buffers includeacetate, adipate, benzoate, citrate, lactate, maleate, phosphate,tartarate and the salts of various amino acids. See Wang (1980) at page455. Suitable stabilizers include carbohydrates such as threlose orglycerol. Suitable reducing agents, which maintain the reduction ofreduced cysteines, include dithiothreitol (DTT also known as Cleland'sreagent) or dithioerythritol at 0.01% to 0.1% wt/wt; acetylcysteine orcysteine at 0.1% to 0.5% (pH 2-3); and thioglycerol at 0.1% to 0.5% (pH3.5 to 7.0) and glutathione. See Akers (1988) at pages 225 to 226.Suitable antioxidants include sodium bisulfite, sodium sulfite, sodiummetabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, andascorbic acid. See Akers (1988) at pages 225. Suitable chelating agents,which chelate trace metals to prevent the trace metal catalyzedoxidation of reduced cysteines, include citrate, tartarate,ethylenediaminetetraacetic acid (EDTA) in its disodium, tetrasodium, andcalcium disodium salts, and diethylenetriamine pentaacetic acid (DTPA).See e.g., Wang (1980) at pages 457-458 and 460-461, and Akers (1988) atpages 224-227. Suitable sugars include glycerol, threose, glucose,galactose and mannitol, sorbitol. A suitable protein is human serumalbumin.

In liquid form, a typical unit dose pharmaceutical composition of thepresent invention comprises from about 5 ng to less than 135,000 ng ofan angiogenic agent dissolved in from 0.1 ml to 10 ml of apharmaceutically acceptable carrier. Because the pharmaceuticalcompositions of the present invention is administered via a cardiaccatheter or other injection device, which has dead space, it isconvenient to formulate the vial containing the pharmaceuticalcomposition so that it contains more of the pharmaceutical compositionthan is to be administered to the patient. For example, when the dose ofthe angiogenic agent to be administered is 45 ng, the vial is formulatedto contain 60-75 ng of angiogenic agent with the excess solutionsuitable for filling the dead space in the delivery equipment. In analternative embodiment that does not allow for dead space, thepharmaceutical composition is loaded in the cardiac catheter in front ofa pharmaceutically acceptable buffer, diluent or carrier, which is thenused to deliver the appropriate amount of the one or more dosages to theone or more sites in the myocardium that are in need of angiogenesis. Asdiscussed above, the pharmaceutically acceptable carrier for theabove-described pharmaceutical compositions comprises a buffer and oneor more stabilizers, reducing agents, anti-oxidants and/or anti-oxidantchelating agents.

When the angiogenic agent is an FGF and the pharmaceutically acceptablecarrier is a liquid carrier, a typical pharmaceutical compositioncomprises about 5 to about 135,000 ng/ml, more typically 5 to 67,500ng/ml, of an FGF or an angiogenically fragment or mutein thereof, 10 mMthioglycerol, 135 mM NaCl, 10 mM sodium citrate, and 1 mM EDTA, pH 5. Asuitable diluent or flushing agent for the above-described compositionis any of the above-described carriers. Typically, the diluent is thecarrier solution itself, which in this example comprises 10 mMthioglycerol, 135 mM NaCl, 10 mM sodium citrate and 1 mM EDTA, pH 5.

When provided in liquid form, the unit dose pharmaceutical compositionsof the present invention become unstable when stored for extendedperiods of time. To maximize stability and shelf life, the unit dosepharmaceutical compositions of the present invention should be storedfrozen at −60° C. When thawed, the solution is stable for 6 months atrefrigerated conditions. A typical vial of the unit dose pharmaceuticalcomposition of the present invention would comprise about 1.0 to 100 ml(more typically, about 1.0 to 25 ml; most typically, about 1.0 to 10 ml)of the above described pharmaceutically acceptable carrier containingtherein from about 5 ng to less than 135,000 ng of an angiogenic agentor an angiogenically fragment or mutein thereof.

In another embodiment, the unit dose pharmaceutical composition of thepresent invention is provided in lyophilized (freeze-dried) form. Inlyophilized form, the unit dose pharmaceutical composition would becapable of being stored at refrigerated temperatures for substantiallylonger than 6 months without loss of therapeutic effectiveness.Lyophilization is accomplished by the rapid freeze drying under reducedpressure of a solution comprising an effective amount of the angiogenicagent dissolved in a pharmaceutically acceptable carrier. Lyophilizers,which perform the above-described lyophilization, are commerciallyavailable and readily operable by those skilled in the art. Typically, aplurality of vials, each containing therein a pharmaceutical composition(containing one or more doses) or a unit dose composition of the presentinvention are placed in a lyophilizer in batch and subjected to coolingand reduced pressure until all liquid carrier is removed. Prior toadministration to a patient, the lyophilized product is reconstituted toa known concentration, preferably in its own vial, with an appropriatesterile aqueous diluent, typically 0.9% (or less) sterile salinesolution, or some other pharmaceutically acceptable carrier. Dependingupon the need for angiogenesis as assessed by the treating physician, aunit dose comprising from 5 ng to less than 135,000 ng, typically fromabout 5 ng to about 67,500 ng, of an angiogenic agent are administeredas a single injection or as a series of injections, typically from 2 to40 injections, into the ischemic myocardium in need of angiogenesis.

In its third aspect, the present invention is directed to a method forinducing angiogenesis (or increasing vascular perfusion, or increasingvascular density or increasing regional myocardial function as measuredby DSE) in a heart of a patient, comprising administering an effectiveamount of an angiogenic agent directly into the myocardium of saidpatient in one or more areas in need of angiogenesis, the effectiveamount of angiogenic agent being from about 5 ng to less than 135,000 ngof said angiogenic agent. Typically, the effective amount of angiogenicagent is from 5 ng to 67,500 ng of said angiogenic agent. Preferably,the patient is a human patient. More preferably, the human patient hassymptoms of coronary artery disease (CAD) or a myocardial infarction(MI). The terms “vascular perfusion” and “vascular density,” asreferenced above, are objective measures of angiogenesis. Increases in“vascular perfusion” and “vascular density” in response to administeringan angiogenic agent according to the method of the present invention areshown in FIGS. 4 and 6-8 herein. Increases in regional cardiac functionproduced by administering unit doses of an angiogenic agent inaccordance with the method of the present invention are shown in FIGS. 5and 11.

In the above-described method, the angiogenic agent is a member selectedfrom the group PDGF, VEGF-A, VEGF-D, TGF-β1, FGF, or an angiogenicallyactive mutein or fragment thereof. Preferably, the angiogenic agent isVEGF-A, VEGF-D or an FGF or an angiogenically active fragment or muteinthereof. More preferably, the angiogenic agent is an FGF, such as FGF-1,FGF-2 or FGF-5, or an angiogenically active fragment or mutein thereof.Most preferably, the angiogenic agent is FGF-2, or an angiogenicallyactive fragment or mutein thereof.

In the above-described method, the angiogenic agent is delivered to themyocardium of a patient in need of angiogenesis using any one of the artknown techniques for myocardium drug delivery. The need of a patient forangiogenesis is evaluated by the treating physician using conventionalevaluation techniques such as coronary angiography, MRI and the like. Inits simplest embodiment, a needle attached to a drug delivery device,such as a syringe, is stereotactically directed from outside the bodythrough the chest cavity and the pericardium to an area of themyocardium in need of angiogenesis for delivery therein of an effectiveamount of an angiogenic agent. Once a dosage has been delivered to themyocardium, the needle is withdrawn or repositioned to one or more siteson the myocardium for delivery of the angiogenic agent. Regardless ofthe number of injection sites in the myocardium (typically, 2-40), thetotal amount of angiogenic agent that is delivered is within the rangeof about 5 ng to less than 135,000 ng, more typically, from 5 ng to67,500 ng. Because the myocardium contracts after delivery of theangiogenic agent, it is believed that some small amount of the dose ofangiogenic agent would be forced back out of the myocardium, via theneedle hole, and into the pericardial space, where momentarily, it wouldprovide a localized concentration at the area of need, and subsequentlyupon mixing in the pericardial fluid, it would continue to bathe themyocardium in angiogenic agent for a prolonged period of time. Theseeffects would only serve to enhance the effect of the IMc dose of theangiogenic agent of the present invention. Thus, in another aspect, thepresent invention is directed to a method for inducing angiogenesis inthe heart of a patient, comprising administering a unit dose ofangiogenic agent directly into the myocardium of a patient in need ofangiogenesis and allowing a residual amount of said angiogenic agent toenter into the pericardial space surrounding said myocardium.

In another embodiment of the method for inducing angiogenesis (orincreasing vascular perfusion, or increasing vascular density orincreasing regional myocardial function as measured by DSE), the unitdose of angiogenic agent is delivered directly into the myocardium froma device having its proximal end outside the body and its distal endpositioned within a coronary vein, a coronary artery or a chamber of theheart. A plurality of devices for delivering medicaments by injectioninto the myocardium from a coronary vein, coronary artery or from achamber of the heart are well known in the art. Examples of such devicesinclude cardiac catheters having a retractable needle at the distal end,which upon being positioned adjacent an area of the myocardium in needof angiogenesis, can project the needle into the myocardium for deliveryof a predetermined amount of medicament. In the present method, such adevice delivers an ultra-low dose of angiogenic agent of the presentinvention to an area of the myocardium in need of angiogenesis. Afterdelivery of the angiogenic agent, the needle is retracted into thedistal end, and the distal end of the device is repositioned adjacent asecond area of the myocardium in need of angiogenesis, whereupon theneedle is again projected into the myocardium and an ultra-low dose ofthe angiogenic agent is delivered. This procedure is then repeated asoften as needed. The needle of the above-described embodiment is alsoreplaceable by a laser, such as used in laser angioplasty, wherein thelaser is used to bore a channel into the area of the myocardium in needof angiogenesis, and an orifice adjacent the laser delivers theultra-low dose of the angiogenic agent directly into the channel. Thislatter device is described in WO 98/05307, entitled “Transmural DrugDelivery Method and Apparatus,” and in corresponding U.S. Ser. No.08/906,991, filed Aug. 6, 1997, and assigned to LocalMed, Palo AltoCalif. Similar cardiac catheters suitable for drug delivery arecommercially available from manufacturers such as ACS, Guidant, Angion,and LocalMed.

Other devices that are suitable for delivery of a medicament to themyocardium include delivery devices having a series of drug deliverypores positioned on the outer surface of the balloon portion of aconventional balloon cardiac catheter, which upon inflating the balloon,bring the drug delivery pores in direct contact with the vascularepithelium. The medicament is then delivered through the drug deliverypores under pressure which forces the medicament past the epithelium andinto the underlying myocardium. Devices of this type are disclosed inU.S. Pat. No. 5,810,767, entitled “Method and Apparatus for PressurizedIntraluminal Drug Delivery” which issued on Sep. 22, 1998; and in U.S.Pat. No. 5,713,860, entitled “Intravascular Catheter with InfusionArray” which issued on Feb. 3, 1998; and in pending application WO97/23256, entitled “Localized Intravascular Delivery of Growth Factorsfor Promotion of Angiogenesis” and corresponding U.S. Ser. No.08/753,224, now pending.

The above-described cardiac catheters are utilized using standardtechniques for cardiac catheter use. Typically, the treating physicianinserts the distal end of the catheter into the femoral or subclavianartery of the patient in need of coronary angiogenesis, and whilevisualizing the catheter, guides the distal end into a coronary artery,vein or chamber of the heart that is proximate to the area of the heartin need of angiogenesis. The distal end of the catheter is positionedadjacent an area of the myocardium in need of angiogenesis and used asdescribed above to deliver an ultra-low dose, i.e., an angiogenicallyeffective amount, of an angiogenic agent. In accordance with the presentinvention, an angiogenically effective amount of an angiogenic agentcomprises from about 5 ng to less than 135,000 ng, typically from 5 ngto 67,500 ng, of the angiogenic agent. Although an angiogenicallyeffective amount of the angiogenic agent is injected into the myocardiumwith each repositioning of the delivery device, the total amount ofangiogenic agent that is injected is less than 135,000 ng (i.e., lessthan 135 μg).

In other embodiments of the above-described method, one or more doses ofthe angiogenic agent are administered to the appropriate areas ofmyocardium for several days, over a series of alternating days, forweeks or over a series of alternating weeks. However, the total amountof angiogenic agent that is injected in one treatment regime is lessthan 135,000 ng (i.e., less than 135 μg).

The diseases most often associated with a need for coronary angiogenesisare coronary artery disease (CAD), i.e., a disease in which one or morecoronary arteries in the patient have become partially occluded, andmyocardial infarction (MI), i.e., a disease in which a coronary arteryhas become sufficiently occluded to cause the necrosis of the downstreammyocardial tissue that relied on the artery for oxygenated blood. Thusin another aspect, the present invention is also directed to a methodfor treating a patient for CAD or MI, comprising administering aneffective amount of an angiogenic agent directly into the myocardium ofsaid patient in one or more areas in need of angiogenesis, the effectiveamount of angiogenic agent being from about 5 ng to less than about135,000 ng of said angiogenic agent. Typically, the effective amount ofangiogenic agent is from about 5 ng to about 67,500 ng of saidangiogenic agent. Preferably, the patient is a human patient.

The active agent in the Applicants' above described pharmaceuticalcomposition, unit dose, or methods is preferably a recombinant FGF or anangiogenically active fragment or mutein thereof. More preferably, theangiogenic agent is FGF-2 or an angiogenically active fragment or muteinthereof.

Clinical efficacy of the ultra-low dose of angiogenic agent of thepresent invention was established in a series of steps whereinangiogenic agent was administered to animals and humans in decreasinglysmaller amounts. The angiogenic agent of these clinical studies wasrecombinant mature bFGF-2 having 146 residues, as disclosed in U.S. Pat.No. 4,956,455 (Baird), and referred to hereinafter as rbFGF-2. Aspreliminary evidence of the clinical efficacy of the ultra-low dosagesof angiogenic agents used herein, human patients exhibiting symptoms ofsevere CAD, who remained symptomatic despite optimal medical management,were administered decreasing dosages of rbFGF-2 by intracoronaryinfusion via a cardiac catheter. TABLE 2 COMPARISON OF QUALITY OF LIFEBEFORE AND 57 DAYS AFTER IC FGF-2 SEATTLE ANGINA QUESTIONNAIRE (SAQ)BASELINE (PRE FGF-2) 57 DAYS POST FGF-2 SUBSCALES MEAN SCORE ± SD MEANSCORE ± SD MEAN CHANGE¹ p VALUE n Exertional Capacity 55 ± 23 68 ± 2513* 0.02 28 Angina Frequency 42 ± 32 66 ± 28 24* <0.001 28 AnginaStability 46 ± 26 82 ± 20 36* <0.001 27 Disease Perception 40 ± 21 61 ±26 19* <0.001 28 Treatment Satisfaction 74 ± 24 88 ± 16 14* 0.002 28*Significantly different from baseline to fifty-seven days.¹A mean change of 8 points or more is considered clinically significant.

TABLE 3 IMPROVEMENTS IN THE QUALITY OF LIFE AT DAY 57 (POST IC rFGF-2)AT LOWER AND HIGHER DOSES SEATTLE ANGINA DOSE <2 μg/kg IC DOSE >2 μg/kgIC QUESTIONNAIRE rFGF-2 (n = 7) rFGF-2 (n = 8) INDEPEN- (SAQ) SUBSCALESMean Change In Score Mean Change In Score DENT Subscales (Day 57Score-Screen Score) (Day 57 Score-Screen Score) SAMPLES T-TESTExertional Capacity 12.30 (23.3) 15.98 (28.7) t = −.27 p = .79 DiseasePerception 26.19 (26.9) 24.47 (21.2) t = .14 p = .89 TreatmentSatisfaction 22.32 (27.7) 10.93 (17.3) t = .97 p = .35 Angina Frequency28.57 (27.3) 13.75 (22.6) t = 1.15 p = .27 Angina Stability 58.13 (12.9)32.14 (34.5) t = 1.75 p = .1081. Possible range for each subscale is 0 to 100 with higher scoresindicating better quality of life.2. Standard deviation noted in parentheses.

TABLE 4 MEAN DATA AND RESULTS AS A FUNCTION OF TIME AND DOSE BASELINE 30DAY 60 DAY Angina Class 2.6 ± 0.7  1.4 ± 0.9 ***  1.2 ± 0.8 *** ExerciseTime (min.) 8.5 ± 2.6  9.4 ± 1.9 *** 10.0 ± 2.5 ** LV EF (%) 47.4 ± 12.347.4 ± 10.6 48.6 ± 11.0 Target Wall 15.4 ± 10.1 23.5 ± 12.0 * 24.1 ±10.1 ** Motion (%) Target Wall 28.7 ± 14.0 34.7 ± 14.1 45.9 ± 11.7 **Thickening (%) Delayed Arrival 18.9 ± 8.3   7.1 ± 3.6 *** 1.82 ± 2.4 ***Zone (% LV)* = p < 0.05** = p < 0.01*** = p < 0.001 (2-tailed, paired)(See Example 3) The doses of FGF-2 administered (and number of patients)were 0.33 μg/kg (n=4), 0.65 μg/kg (n=4), 2.0 μg/kg (n=8), 6.0 μg/kg(n=4), 12.0 μg/kg (n=4), 24 μg/kg (n=8), 36 μg/kg (n=10) and 48 μg/kg(n=10). Angina frequency and quality of life was assessed by the SeattleAngina Questionnaire (SAQ) at a baseline (before FGF-2 administration)and at about 60 days after FGF-2 administration. Exercise tolerance time(ETT) was assessed by the threadmill test. Rest/exercise nuclearperfusion and gated sestamibi-determined rest ejection fraction (EF),and magnetic resonance imaging (MRI) were assessed at baseline, and at30 days and 60 days post FGF-2 administration. Other end points thatwere evaluated included MRI (to objectively measure ejection fraction(EF), normal wall motion (NWM), targeted wall motion (TWM), normal wallthickness (NWT), targeted wall thickness (TWT), ischemic area zone andcollateral extent). See Tables 2-4, respectively. The patients exhibitedsignificant clinical improvements to all dosages of the FGF-2 that wereadministered IC. In particular, Table 3 discloses that the patientsreceiving the lowest dosages of FGF-2 (less than 2 μg/kg) exhibitedbetter results in four of the five criteria assessed than did thepatients receiving the higher dosages of FGF-2 (greater than 2 μg/kg).The above described method for treating CAD, when assessed by thestandard objective criterion employed in the art (i.e., ETT), providedan unexpectedly superior increase of one and a half to two minutes inthe treated patient's ETT. This compares exceptionally well whencompared to the increase of 30 seconds that is deemed clinicallysignificant for the current mode of treatment, i.e., angioplasty.

A major side effect reported in the art for the angiogenic agents of thepresent invention is acute hypotension. This is due to the known effectof many of the angiogenic agents as a vasodilator. However, no adversehypotensive effects were observed following administration, alone or inseries, of any of the ultra-low dosages of angiogenic agent within thescope of the present invention.

In testing the angiogenic agents for angiogenic activity in vivo,fifty-two (52) human patients diagnosed with CAD, who satisfied thecriteria of Example 2 herein, were administered a unit dose of 0.33μg/kg to 48 μg/kg of the FGF-2 by intracoronary (IC) infusion over abouta 20 minute period. In particular, in the 52 patients, a coronary(cardiac) catheter was inserted into an artery (e.g., femoral orsubclavian) of the patient in need of treatment and the catheter waspushed forward with visualization, until it was positioned in theappropriate coronary artery of the patient to be treated. Using standardprecautions for maintaining a clear line, the angiogenic agent wasadministered by infusing the unit dose substantially continuously over aperiod of 10 to 30 minutes. The 52 treated patients were then assessedby the Seattle Angina Questionnaire, which provides an assessment basedupon a mixed combination of objective and subjective criteria. See Table2. The Seattle Angina Questionnaire is a validated, disease-specificinstrument with the following five subscales that are assessed bothbefore and after treatment: 1) “exertional capacity”=limitation ofphysical activity; 2) “disease perception”=worry about MI; 3) “treatmentsatisfaction”; 4) “angina frequency”=number of episodes and sublingualnitroglycerin usage; and 5) “angina stability”=number of episodes withmost strenuous physical activity. The possible range for each of thefive subscales is 0 to 100 with the higher scores indicating a betterquality of life. Moreover, a mean change of 8 points or more between themean baseline scores (before treatment) and the post-treatment scores isrecognized as being “clinically significant.” Table 2 reports that the28 patients, who were pretested and then administered a single unit doseof 0.33 μg/kg to 24 μg/kg of rbFGF-2 by IC infusion, exhibited a meanscore increase of 13 to 36 points for the five “quality of life”criteria assessed by the “Seattle Angina Questionnaire.” See Table 2herein. These 13 to 36 point increases were about 1.6 to 4.5 timesgreater than the 8 point change which is recognized in the art as being“clinically significant” in alternative modes of treatment. See Table 2herein. Moreover, when the combined results for the first 15 patients ofTable 2 were broken down between low dose (less than or equal to 2μg/kg) and high (more than 2 μg/kg) doses of rbFGF-2, and assessed bythe “Seattle Angina Questionnaire,” both doses were found to provideincreased scores that ranged from about 12.3 to 58.1 and about 10.9 to32.1, respectively. See Table 3 herein. The increased scores were about1.4 to 7.2 times greater than the 8 point change which is considered tobe “clinically significant” in alternative modes of treatment.

In the same Phase I trial, fifty-two human patients who were diagnosedwith CAD and who satisfied the criteria of Example 2 herein, wereadministered IC a single unit dose of 0.33 μg/kg to 48 μg/kg of rbFGF-2.The maximum tolerated dose was defined as 36 μg/kg by severe buttransient hypotension that was observed in 2 out of 10 patients at thenext higher dose of 48 μg/kg. At one of the sites, the hearts of 23patients were assessed both before (“baseline”) and 30 and 60 days aftertreatment by magnetic resonance imaging (MRI) for objective signs ofimproved coronary sufficiency. Among the objective criteria assessed byMRI are the following: 1) left ventricular (LV) ejection fraction (EF);2) normal wall thickness (NWT); 3) normal wall motion (NWM); 4)collateral extent; 5) ischemic area zone; 6) targeted wall thickness(TWT); 7) targeted wall motion (TWM); and 8) perfusion or delayedarrival zone (% LV). The patients were also assessed for angina,treadmill exercise duration, rest/exercise nuclear perfusion. Theresults are summarized in Table 4. Table 4 reflects that the baselineangina class decreased from 2.6 to 1.4 and 1.2 at 30 and 60 days,respectively post IC FGF-2. The mean treadmill exercise time increasedfrom a baseline of 8.5 minutes to 9.4 and 10.0 minutes at 30 and 60days, respectively, post treatment. No significant difference wasobserved in the left ventricular ejection fraction (LV EF). However, thetarget wall motion increased significantly, moving from a baseline of15.4% to 23.5% (day 30) and 24.1% (day 60) post FGF-2 treatment.Likewise the target wall thickening increased significantly from abaseline of 28.7% to 34.7% (day 30) and 45.9% (day 60) post FGF-2treatment. There was also a significant increase in perfusion, asmeasured by a decrease in the delayed arrival zone (% LV), with thedelayed arrival zone decreasing from a baseline of 18.9% to 7.1% (day30) and 1.82% (day 60) post FGF-2 treatment. Thus, providing CADpatients with a single IC infusion of an angiogenic agent, such asFGF-2, provided the patients with a significant physical improvement asobjectively measured by MRI and other conventional criteria.

Pharmacokinetics and Metabolism

The kidneys and liver are the major organs for the elimination of theangiogenic agents. In particular, the kidneys have a protein cutoff ofabout 60 kD and thus retain serum albumin (MW 60 kD). However, all theangiogenic agents of the present invention have a molecular weight lessthan 40 kD. FGF-2, the angiogenic agent of the present Examples, has amolecular weight of about 16 kD. Accordingly, renal excretion is to beexpected. In a radiolabelled biodistribution study of commerciallyavailable bFGF-2, both the liver and the kidney were shown to containhigh counts of the radiolabelled bFGF-2 at 1 hour after IV or ICinjection. In a published study, wherein another recombinant iodinatedform of bFGF-2 was given to rats, the liver was identified as the majororgan of elimination. Whalen et al., “The Fate of IntravenouslyAdministered bFGF and the Effect of Heparin,” Growth Factors, 1:157-164(1989). More particularly, it is known that FGF-2 binds in the generalcirculation to α₂-macroglobulin and that this complex is internalized byreceptors on the Kupffer cells. Whalen et al. (1989) and LaMarre et al.,“Cytokine Binding and Clearance Properties of Proteinase-ActivatedAlpha-2-Macroglobulins,” Lab. Invest., 65:3-14 (1991). Labelled FGF-2fragments were not found in the plasma, but they were found in the urineand corresponded in size to intracellular breakdown products. When FGF-2was administered in combination with heparin, the renal excretion ofFGF-2 was increased. Whalen et al. (1989). The FGF-2 molecule, which iscationic when not complexed with heparin, is likely repelled by thecationic heparin sulfate of the glomerular basement membrane. TheFGF-2/heparin complex is more neutrally charged, and therefore is moreeasily filtered and excreted by the kidney.

The pharmacokinetics of FGF-2 were determined after intravenous (IV) andintracoronary (IC) administration in domestic Yorkshire pigs, after IVdosing in Sprague Dawley (“SD”) rats, and after IC administration in CADhuman patients. In all species, the rFGF-2 plasma concentrations afterIV and/or IC injection followed a biexponential curve with an initialsteep slope and considerable decrease over several log scales (thedistribution phase) during the first hour, followed by a more moderatedecline (the elimination phase). FIG. 1 provides a plasma concentrationversus time curve showing these phases in humans after IC administrationof recombinant mature bFGF-2 (146 residues) as a function of thefollowing doses: 0.33 μg/kg, 0.65 μg/kg, 2 μg/kg, 6 μg/kg, 12 μg/kg and24 μg/kg of lean body mass (LBM). The plasma concentrations of bFGF-2were determined by a commercially available ELISA (R&D Systems,Minneapolis Minn.) that was marketed for analysis of human FGF-2. TheELISA assay for hFGF-2 showed 100% cross-reactivity with the recombinantmature bFGF-2. Other members of the FGF family, as well as many othercytokines, were not detected by this assay. Also, heparin does notinterfere with the assay.

The design of these pharmokinetic studies, pharmacokinetic parameters,and conclusions are listed in Tables 5 and 6 for studies in pigs andrats, respectively. The reader is referred to these tables for thespecific details. However, among the points to be noted are that thehalf-life (T_(1/2)) was 2.8±0.8 to 3.5 hours following a single ICinfusion for the single component model for animals having a clearance(CL) of 702±311 to 609±350 ml/hr/kg. The results of this study TABLE 5PHARMACOKINETICS (PK) AND PHARMACODYNAMICS OF rFGF-2 IN PIGS ANIMALSDOSING REGIMEN PK PARAMETERS RESULTS Domestic Yorkshire Pigs 2-20 μg/kgIV bolus CL = 702 ± 311 mL/hr/kg Systemic PK identical under generalanesthesia 2-20 μg/kg IC bolus T½ = 2.8 ± 0.8 hr. between IV and ICroute (n = 13; 30 ± 5 kg) 20 μg/kg by 10 min IC infusion Fastdistribution phase 70 U/kg heparin ˜ 15 Dose-linearity min before rFGF-2Transient decreases of MAP Domestic Yorkshire Pigs 0.65-6.5 μg/kg by5-mm CL = 609 ± 350 ml/hr/kg No gender difference in PK under generalanesthesia IC infusion T½ = ˜3.5 hr Biphasic decline of (n = 17; 26 ± 4kg) 70 U/kg heparin ˜ 15 min 3-Comp. Model: plasma rFGF-2 before rFGF.2T½ α = 1.5 min Dose-linearity T½ β = 17 min V₁ equal T½ γ = 6.6 hr to ˜plasma volume CL = 580 ml/hr/kg V₅₆ equal to ˜ 10-fold V₁ = 55 ml/kgplasma volume V₅₆ = 523 ml/kg Magnitude and duration of MAP decreasecorrelated with rFGF-2 dose and peak plasma level Domestic YorkshirePigs 6.5 μg/kg weekly Without Heparin The rFGF-2 distribution undergeneral anesthesia by 5 min (Doses 1-6): phase was less steep, the (n =6; 25 ± 5 kg) IV infusion for T½ = 2-6 hr volume of distribution 6 weeksCL = 777-2749 ml/hr/kg smaller, and clearance was 70 U/kg heparin 10 minV₅₆ = 871-12,500 ml/kg slower with heparin- before rFGF-2 With Heparinpretreatment (n = 3), or rFGF-2 (Doses 1-6): Binding of rFGF-2 to alone(n = 3) T½ = 2-3 hr circulating heparin appears CL = 235-347 ml/hr/kg todecrease biodistribution V₅₆ = 71-153 ml/kg and elimination Both volumeand clearance of rFGF-2 increased at later doses (potential receptorupregulation), but more so in the absence of heparin Magnitude andduration of MAP decreases were similar with or without heparin

TABLE 6 PHARMACOKINETICS (PK) OF rFGF-2 IN RATS ANIMALS DOSING REGIMENPK PARAMETERS RESULTS Conscious SD rats 3-100 μg/kg bolus IV T½ = 1.1 ±0.51 hr Fast distribution phase (n = 18; 322 ± 93 g) injection CL = 4480± 2700 ml/hr/kg Apparent dose-linearity V₅₅ = 1924 ± 1254 ml/kgConscious SD rats 30-300 μg/kg weekly by T½ = 1.4 ± 0.13 hrTime-invariant PK; (n − 54; 149 ± 12 g) bolus IV injection for 6 CL =1691 ± 169 ml/hr/kg plasma profiles, PK weeks V₅₅ = 1942 ± 358 ml/kgparameters and AUCs were No heparin pretreatment similar over time Doselinearity Conscious SD rats 30 μg/kg bolus IV Time-Averaged PKParameters: In all cases, heparin (27 males; 381 ± 48 g; injection T½ CLV₅₅ increased the rFGF-2 20 females; 268 ± 22 g) No heparin hr. ml/hr/kgml/kg plasma levels 40 U/kg IV Heparin: 0.75 4332 2389 Both volume ofdistribution and at ˜ 15 min 0.91 1728 844 clearance of rFGF-2 were justprior to rFGF-2 1.3 516 147 smaller with heparin at +15 min 1.2 1158 626Greatest changes on CL and V₅₅ at +3 hr. 0.93 1338 1351 were observedwhen heparin was administered immediately prior to rFGF-2show that the pharmacokinetics of the rFGF-2 were substantiallyidentical regardless of whether the animals were dosed via the IC or IVroutes. See Table 5. Among the other pharmacokinetic results to be takenfrom Tables 5 and 6 of these studies is that there is a fastdistribution phase followed by a more moderate elimination phase, anddose linearity as reported in FIG. 1 for humans. Also, there were nogender differences. Further, the three compartment model was analyzedfor pigs receiving 70 U/kg of heparin approximately (“˜”) 15 minutesbefore receiving 0.65-6.5 μg/kg by 5-10 minute IC infusion. The halflives (T_(1/2α), T_(1/2β) and T_(1/2γ)) for the three compartments were1.5 minutes, 17 minutes, and 6.6 hours, respectively. In these animals,the initial volume (“V₁”) was approximately the plasma volume, and thesteady state volume (“V_(ss)”) was approximately 10-fold the plasmavolume. See Table 5. In pigs, the binding of rFGF-2 to circulatingheparin appears to decrease biodistribution and elimination. Likewise,in rats, both the volume of distribution and the clearance of rFGF-2were smaller when heparin was administered. See Table 6. Further, thegreatest and most favorable changes on clearance of FGF-2 were foundwhen heparin was administered within ±15 minutes, preferably immediatelyprior to rFGF-2 IC infusion. See Table 6.

The pharmacokinetics of the FGF-2 was studied in humans, diagnosed withCAD despite optimal medical management, in a Phase I clinical studysupporting this filing. The doses of rbFGF-2 employed in that Phase Istudy were 0.33 μg/kg, 0.65 μg/kg, 2 μg/kg, 6 μg/kg, 12 μg/kg, and 24μg/kg of lean body mass (LBM), and all doses were administered by a 20minute IC infusion (10 minutes into each of two patent coronary vessels)after pretreating the patient with 40 U/kg heparin which wasadministered IV or IC₁₋₉₅ minutes before rbFGF-2 infusion. FIGS. 1-3herein summarize the data underlying those results. In particular, FIG.1 is a plot of the mean FGF-2 plasma concentration versus time (hours)for the six different doses of rbFGF-2 administered by IC infusion asdescribed above over a 20 minute period. FIG. 1 shows dose linearity anda biphasic plasma level decline, i.e., a fast distribution phase duringthe first hour, followed by an elimination phase with T_(1/2) of 1.9±2.2hours. The dose linearity is more readily seen in FIG. 2, which is aplot of the individual patient FGF-2 area under the curve (AUC) inpg·hr/ml for FIG. 1 for each of the six doses of rbFGF-2 administered.FIG. 3 is a plot individual human patient FGF-2 dose normalized AUCsversus time of heparin dose in “minutes prior to rFGF-2 infusion” andshows the influence of timing of heparin administration on FGF-2 AUC.FIG. 3 shows that the greatest AUC/dose was achieved when an effectiveamount of a glycosoaminoglycan, such as heparin, was preadministeredwithin 30 minutes or less of IC rFGF-2 infusion, more preferably within20 minutes or less of IC rFGF-2 infusion. Typically, an effective amountof a glycosoaminoglycan is 40-70 U/kg heparin. These pharmacokineticresults are summarized in Table 7 herein.

The rFGF-2 distribution phase was less steep with heparin, the volume ofdistribution smaller, and the clearance slower, as compared to rFGF-2without heparin. It appears that the complex of rFGF-2 with circulatingheparin decreases the biodistribution and elimination of rFGF-2.Although the binding of FGF-2 to heparin-like structures is strong(dissociation constant ˜2×10⁻⁹ M), the binding of FGF-2 to the FGF-2receptor is approximately two orders of magnitude higher (dissociationconstant ˜2×10⁻¹¹ M). Moscatelli et al., (1991). In addition, thecomplexation of the rFGF-2 with a glycosoaminoglycan, such as a heparin,might increase signal transduction and mitogenesis, and/or protect therFGF-2 from enzymatic degradation. Using a validated and art-acceptedmodel of hibernating myocardium, ten (10) miniswine underwent 90% leftcircumflex (LCx) coronary stenosis. For validation, see e.g.,Yanagisawa-Miwa, et al., “Salvage of Infarcted Myocardium byAngiogenesic Action of Basic Fibroblast Growth Factor,” Science,257:1401-1403 (1992); Banai et al., “Angiogenic-Induced Enhancement ofCollateral Blood Flow to Ischemic Myocardium by Vascular EndothelialGrowth Factor in Dogs,” Circulation, 89(5):2183-2189 (May 1994); andUnger, et al., “Basic fibroblast growth factor enhances myocardialcollateral flow in a canine model,” Am. J. Physiol., 266 (Heart Circ.Physiol. 35): H1588-H1595 (1994). One month later, a baseline positronemission tomography (PET) and dobutamine stress echocardiography wereperformed on the animals. The animals were then randomized and treatedwith 30 injections in the LCx region of either 100 μl carrier (n=5) orrbFGF-2 (45 ng/injection; total dose 1,350 ng) (n=5) in carrier. In theabove injections, the carrier was a sterile aqueous solution comprising10 mM thioglycerol, 135 mM NaCl, 10 mM sodium citrate and 1 mM EDTA, pH5. In the five animals that received the injections of FGF-2 in theirmyocardium, the LCx region myocardial blood flow (MBF) at rest, asmeasured by PET, increased from 61.3±4.4% of non-ischemic septal valuesat baseline (day 0) to 82.8±3.1% at 6 months postoperatively (p<0.001).The wall motion score index (WMSI) at rest for the LCx region was2.4±0.2 at baseline and improved to 2.2±0.2 (p=0.08 vs baseline) at sixmonths. Likewise, WMSI for the LCx region at peak stress was 2.2±0.4 atbaseline (day 0) and decreased to 1.8±0.3 (p=0.05) at six months. Therewas no significant change in MBF or in the resting or stress WMSI in thevehicle treated animals at any time point. Western blot analysis oftissue samples taken from the treated chronically ischemic regionsrevealed significantly greater upregulation of VEGF in the chronicallyischemic regions treated with rFGF-2 versus that observed in thechronically ischemic regions treated with vehicle (p<0.05).

Thus, in this validated model of a patient in need of angiogenesis, thedirect intramyocardial injection of ultra-low dose of angiogenic agent,such as FGF-2, improved MBF and contractile reserve in the treatedregions of the myocardium. Accordingly, an ultra-low dose of angiogenicagent represents a viable method for inducing angiogenesis and a viablealternative therapy for the treatment of CAD and/or MI.

Examples 1-6, which follow, provide more details on the selectioncriterion and on the Phase I clinical trial for IC FGF-2 that gave riseto the preliminary data discussed above. Example 7 discloses data on theultra-low dose pharmaceutical composition and method of the presentinvention and its use to induce coronary angiogenesis in patients(miniswine) in a model system for coronary artery disease and myocardialinfarction.

EXAMPLE 1 Medium Concentration Unit Dose of rFGF-2 Employed in the PhaseI Clinical Trial

The recombinant mature FGF-2 of U.S. Pat. No. 5,155,214 (Baird) wasformulated as a medium concentration (0.2 μg/kg to about 36 μg/kg) unitdose and pharmaceutical composition and administered to rats, pigs andultimately to humans in the Phase I clinical trial referenced herein.The various formulations are described below.

The Medium Concentration rFGF-2 Unit Dose was provided as a liquid in 3cc type I glass vials with a laminated gray butyl rubber stopper and redflip-off overseal. The rFGF-2 unit dose contained 1.2 ml of 0.3 mg/mlrFGF-2 in 10 mM sodium citrate, 10 mM monothioglycerol, 1 mM disodiumdihydrate EDTA (molecular weight 372.2), 135 mM sodium chloride, pH 5.0.Thus, in absolute terms, each vial (and unit dose) contained 0.36 mgrFGF-2. The vials containing the unit dose in liquid form were stored at2° to 8° C.

The rFGF diluent was supplied in 5 cc type I glass vials with alaminated gray butyl rubber stopper and red flip-off overseal. TherFGF-2 diluent contains 10 mM sodium citrate, 10 mM monothioglycerol,135 mM sodium chloride, pH 5.0. Each vial contained 5.2 ml of rFGF-2diluent solution that was stored at 2° to 8° C.

The Medium Concentration rFGF-2 Pharmaceutical Composition that wasinfused was prepared by diluting the rFGF-2 unit dose with the rFGFdiluent such that the infusion volume is 10 ml. In order to keep theEDTA concentration below the limit of 100 μg/ml, the total infusionvolume was increased to 20 ml when proportionately higher absoluteamounts of FGF-2 were administered to patients with high body weights.

EXAMPLE 2 Selection Criteria for Patients with Coronary Artery Diseasefor Treatment with rFGF-2”

The following selection criteria were applied to Phase I patients withcoronary artery disease, whose activities were limited by coronaryischemia despite optimal medical management, and who were not candidatesfor approved revascularization therapies:

-   -   Inclusion criteria: Subject is eligible if:        -   Male or female, greater than or equal to 18 years of age        -   Diagnosis of coronary artery disease (CAD)        -   Suboptimal candidates for approved revascularization            procedures, e.g., angioplasty, stents, coronary artery            bypass graft (CABG) (or refuses those interventions)        -   Able to exercise at least three minutes using a modified            Bruce protocol and limited by coronary ischemia        -   Inducible and reversible defect of at least 20% myocardium            on pharmacologically stressed thallium sestamibi scan        -   CBC, platelets, serum chemistry within clinically acceptable            range for required cardiac catheterization        -   Normal INR, or if anticoagulated with Coumadin, INR <2.0        -   Willing and able to give written informed consent to            participate in this study, including all required study            procedures and follow-up visits    -   Exclusion criteria: Subject is not eligible if:        -   Malignancy: any history of malignancy within past ten years,            with the exception of curatively treated basal cell            carcinoma.        -   Ocular conditions: proliferative retinopathy, severe            non-proliferative retinopathy, retinal vein occlusion,            Eales' disease, or macular edema or funduscopy by            ophthalmologist: history of intraocular surgery within six            months        -   Renal function: creatinine clearance below normal range            adjusted for age; protein >250 mg or microalbumin >30 mg/24            h urine        -   Class IV heart failure (New York Heart Association)        -   Ejection fraction<20% by echocardiogram, thallium scan, MRI            or gated pooled blood scan (MUGA)        -   Hemodynamically relevant arrhythmias (e.g., ventricular            fibrillation, sustained ventricular tachycardia)        -   Severe valvular stenosis (aortic area <1.0 cm², mitral area            <1.2 cm²), or severe valvular insufficiency        -   Marked increase in angina or unstable angina within three            weeks        -   History of myocardial infarction (MI) within three months        -   History of transient ischemic attack (TIA) or stroke within            six months        -   History of CABG, angioplasty or stent within six months        -   History of treatment with transmyocardial laser            revascularization, rFGF-2, or vascular enodothelial growth            factor (VEGF) within six months        -   Females of child-bearing potential or nursing mothers        -   Any pathological fibrosis, e.g., pulmonary fibrosis,            scleroderma        -   Known vascular malformation, e.g., AV malformation,            hemangiomas        -   Coexistence of any disease which might interfere with            assessment of symptoms of CAD, e.g., pericarditis,            costochondritis, esophagitis, systemic vasculitis, sickle            cell disease        -   Coexistence of any disease which limits performance of            modified Bruce protocol exercise stress test, e.g.,            paralysis or amputation of a lower extremity, severe            arthritis or lower extremities, severe chronic obstructive            pulmonary disease (COPD)        -   Participation in clinical trials of investigational agents,            devices or procedures within thirty days (or scheduled            within sixty days of study drug)        -   Known hypersensitivity to rFGF-2 or related compounds        -   Any condition which makes the subject unsuitable for            participation in this study in the opinion of the            investigator, e.g., psychosis, severe mental retardation,            inability to communicate with study personnel, drug or            alcohol abuse

EXAMPLE 3 Phase I Clinical Study on Recombinant FGF-2 Administered IC toHumans

Recombinant FGF-2 of U.S. Pat. No. 5,155,214 was administered to 52human patients with severe CAD, who remained symptomatic despite optimalmedical management and who refused or were suboptimal candidates forsurgical or percutaneous revascularization, in a Phase I open label,single administration, dose escalation, two-site trial. The drug wasadministered as a single 20 minute infusion divided between two majorsources of coronary blood supply (IC), using standard techniques forpositioning a catheter into the patient's coronary artery (such asalready employed in angioplasty). The doses (μg/kg) of rFGF-2administered were 0.33 (n=4), 0.65 (n=4), 2.0 (n=8), 6.0 (n=4), 12.0(n=4), 24 (n=8), 36 (n=10) and 48 (n=10). Angina frequency and qualityof life was assessed by the Seattle Angina Questionnaire (SAQ) at abaseline (before rFGF-2 administration) and at about 60 days afterrFGF-2 administration. Exercise tolerance time (ETT) was assessed by thethreadmill test. Rest/exercise nuclear perfusion and gatedsestamibi-determined rest ejection fraction (EF), and magnetic resonanceimaging (MRI) were assessed at baseline, and at 30 days and 60 days postFGF-2 administration. Other end points that were evaluated included MRI(to objectively measure ejection fraction (EF), normal wall motion(NWM), targeted wall motion (TWM), normal wall thickness (NWT), targetedwall thickness (TWT), ischemic area zone and collateral extent). SeeTables 2-4, respectively.

The preliminary safety results indicate that serious events were notdose related. Thus far, of the eight dosage groups, there were threedeaths in the lowest dosage groups, i.e., at 0.65 μg/kg (Day 23), at 2.0μg/kg (Day 57) and at 6.0 μg/kg (Day 63). There were sixhospitalizations for acute myocardial infarction (MI) in three patients,i.e., one patient from each of groups 1 (0.33 μg/kg), 3 (2.0 μg/kg) and4 (6.0 μg/kg). One of the three patients accounted for four of the sixhospitalizations for acute MI. There was also one large B cell lymphomathat was diagnosed three weeks after dosing in a patient in group 4. Thepatient died at two months post dosing. Acute hypotension, seen athigher doses during or just subsequent to infusion, was managed byadministration of fluids without need for a vasopressor. The maximumtolerated dose (MTD) in humans was defined as 36 μg/kg. (In contrast, inpigs, the MTD was 6.5 μg/ml.) Doses of rFGF-2 up to 48 μg/kg IC weremanaged in patients with aggressive fluid management, but were nottolerated due to acute and/or orthostatic hypotension in two out of tenpatients. The half-life of the IC infused rFGF-2 was about one hour.

The human patients in this study that were treated with a single ICinfusion of rFGF-2 exhibited a mean increase in ETT of 1.5 to 2 minutes.This is especially significant because an increase in ETT of >30 secondsis considered significant and a benchmark for evaluating alternativetherapies, such as angioplasty. The angina frequency and quality oflife, as measured by SAQ, showed a significant improvement at 57 days inall five subscales for the 28 patients (n=28) tested. See Tables 2 and3. In particular, the mean changes in scores for the five criteriaevaluated by the SAQ ranged from 13 to 36 with a mean change of 8 ormore considered “clinically significant.” See Table 2.

Magnetic resonance imaging (MRI) showed objective improvements followingadministration of a single unit dose of the FGF-2, including increasedtargeted wall motion at 30 and 60 days (p<0.05), and increased targetedwall thickening at 60 days (p<0.01). MRI further showed improvedregional wall motion, and increased myocardial perfusion and collateraldevelopment in the targeted area for both the lower dose (0.33 μg/kg and0.65 μg/kg) and higher dose (2.0 μg/kg and 12.0 μg/kg) groups in an 11patient test group (n=11).

Abnormal perfusion zone, which was assessed at one of the sites on 28patients, decreased significantly at 30 and 60 days (p<0.001).

In addition to the above criterion (i.e., ETT SAQ, MRI), a treatment isconsidered very successful if the angiogenic effects last at least sixmonths. In the present Phase I study, the unexpectedly superiorangiogenic effects were observed to last for 57-60 days in all dosagegroups. [See Tables 2-4.] Based upon the results already obtained, it isexpected that the angiogenic effects would last twelve months or morebut at least six months, at which time the procedure could be repeated,if necessary.

EXAMPLE 4 Proposed Phase II Clinical Study on Recombinant FGF-2Administered to Humans to Treat Coronary Artery Disease

The Phase II clinical trial of rFGF-2 of U.S. Pat. No. 5,155,214 fortreating human patients for coronary artery disease is performed as adouble blind/placebo controlled study having four arms: placebo, 0.3μg/kg, 3 μg/kg and 30 μg/kg administered IC.

EXAMPLE 5 Unit Dose and Pharmaceutical Composition of rFGF-2 for thePhase II Human Clinical Trial

The rFGF-2 of U.S. Pat. No. 5,155,214 was formulated as a stockpharmaceutical composition for administration to humans in the Phase IIclinical trial referenced herein. The various formulations are describedbelow.

The Medium Concentration rFGF-2 stock pharmaceutical composition forExamples 2-4 was prepared as a liquid in 5 cc type I glass vials with alaminated gray butyl rubber stopper and red flip-off overseal. TherFGF-2 composition contained 0.3 mg/ml rFGF-2 of U.S. Pat. No. 5,155,214in 10 mM sodium citrate, 10 mM monothioglycerol, 0.3 mM disodiumdihydrate EDTA (molecular weight 372.2), 135 mM sodium chloride, pH 5.0.Each vial contained 3.7 ml of rFGF-2 drug product solution (1.11 mgrFGF-2 per vial). The resulting FGF-2 stock pharmaceutical compositionin liquid form was stored at 2° to 8° C. Prior to use, theabove-described FGF-2 composition was diluted with the “rFGF-2 placebo.”

The rFGF placebo is supplied as a clear colorless liquid in 5 cc type Iglass vials with a laminated gray butyl rubber stopper and red flip-offoverseal. The rFGF-2 placebo is indistinguishable in appearance from thedrug product and has the following formulation: 10 mM sodium citrate, 10mM monothioglycerol, 0.3 mM disodium dihydrate EDTA (molecular weight372.2), 135 mM sodium chloride, pH 5.0. Each vial contains 5.2 ml ofrFGF-2 placebo solution. Like the unit dose, the rFGF-2 placebo isstored at 2° to 8° C.

The Medium Concentration rFGF-2 pharmaceutical composition that wasultimately infused IC, as described in Examples 2-4 herein, was preparedby diluting the rFGF-2 unit dose with the rFGF diluent such that theinfusion volume is 10 ml. In order to keep the EDTA concentration belowthe limit of 100 μg/ml, the total infusion volume was increased to 40 mlwhen proportionately higher absolute amounts of FGF-2 were administeredto subjects with high body weights.

EXAMPLE 6 Selection Criteria for CAD Patients for the Phase II HumanClinical Trial of IC rFGF-2

Accordingly, the above-described evidence of an unexpectedly superiorimprovement in quality of life and of increased angiogenic efficacy inhumans who were administered a single unit dosage of rFGF-2 inaccordance with this invention, supports the patentability of theApplicants' unit dose, pharmaceutical composition and method of usingthe same.

EXAMPLE 7 Inducing Angiogenesis In Vivo by the Administration ofUltra-low Doses of rFGF-2 to the Myocardium of Miniswine

Using a validated model of hibernating myocardium, miniswine underwent90% left circumflex (LCx) coronary stenosis. Briefly, a hydraulicallycontrolled occluder was placed around the proximal end of the LCx ofminiswine. A flow probe was inserted into the LCx distal to thehydraulic occluder and the occluder was inflated to consistently provide90% occlusion. The animals were tested in groups of 6. One month later,baseline positron emission tomography (PET) and dobutamine stressechocardiography (DSE) were performed and the animals randomized to 30injections of either 100 μl carrier (n=5) or rFGF-2 in carrier (45ng/injection; total dose 1.35 μg) (n=5) in the LCx region. In the aboveinjections, the FGF-2 was the recombinant mature FGF-2 (SEQ ID NO: 2) ofU.S. Pat. No. 5,155,214. The carrier was a sterile aqueous solutioncomprising 10 mM thioglycerol, 135 mM NaCl, 10 mM sodium citrate and 1mM EDTA, pH 5. The total dose (1.35 μg) of FGF-2 provided in thisexample is 1/100 the intracoronary (IC) delivered dose (135 μg) that wasfound to be effective in the ameroid porcine model, wherein the LCx wasoccluded 100%.

In the animals that received the injections of rFGF-2 in theirmyocardium, the LCx region myocardial blood flow (MBF) at rest, asmeasured by PET, increased from 61.3±4.4% of non-ischemic septal valuesat baseline (day 0) to 82.8±3.1% at 6 months postoperatively (p<0.001).The wall motion score index (WMSI) at rest for the LCx region was2.4±0.2 at baseline and improved to 2.2±0.2 (p=0.08 vs baseline) at sixmonths. Likewise, WMSI for the LCx region at peak stress was 2.2±0.4 atbaseline (day 0) and improved to 1.8±0.3 (p=0.05) at six months. (FIG.5) There was no significant change in MBF or rest or stress WMSI in thevehicle treated animals at any time point. Six months after treatment,the miniswine were sacrificed and the capillary density of the treatedischemic myocardium was determined. The FGF-2 treated miniswineexhibited a capillary density of about 4400/unit volume, versus about1700 for the saline treated group. (FIG. 6) Western blot analysisrevealed significantly greater upregulation of VEGF (measured asVEGF₁₆₅) and FGF-2 in the chronically ischemic FGF-2 treated regionsversus that observed with vehicle (p<0.05). FIG. 10. Surprisingly, theupregulation of VEGF and FGF-2 continued for at least 3 months aftertreatment. (FIG. 10). Thus, the direct intramyocardial injection of anultra-low dose of angiogenic agent, such as rFGF-2, improves MBF,contractile reserve, perfusion (FIG. 4), myocardial function as measuredby DSE (FIG. 5), and capillary density (FIG. 6) in the treated regionsof the myocardium. Accordingly, injecting an ultra-low dose ofangiogenic agent IMc represents a viable method for inducingangiogenesis and a viable alternative therapy for the treatment of CADand/or MI.

EXAMPLE 8 Inducing Angiogenesis In Vivo by Administration of VariousDoses of rFGF-2 to the Myocardium of Miniswine

Using the same validated model of hibernating myocardium described inExample 7, miniswine underwent 90% left circumflex (LCx) coronarystenosis. Briefly, a hydraulically controlled occluder was placed aroundthe proximal end of the LCx of miniswine. A flow probe was inserted intothe LCx distal to the hydraulic occluder and the occluder was inflatedto consistently provide 90% occlusion. Four groups of animals weretested in groups of 6. The groups were as follows:

-   -   IMc mid dose: 6 animals @ 0.6 μg/kg total dose IMc        -   30 injections in LCx territory, no heparin IMc    -   IMc high dose: 6 animals @ 6.0 μg/kg total dose IMc        -   30 injections in LCx territory, no heparin IMc    -   Positive control: 6 animals® 6.0 μg/kg I.C. in the ameroid model        (100% occlusion of the LCx) total dose 135 μg, delivered as        -   heparin 70 U/kg 5 min before start of infusion        -   ½ dose RCA if possible, ½ dose LCx or LAD (3 μg/kg/artery),            each delivered by infusion over 10 minutes per artery (20            minutes total infusion time)    -   Negative control: 6 animals−vehicle/saline×30 injections IMc.        The miniswine were randomly assigned to treatment groups at time        of surgery.        Phase 1: Establishment of Baseline and Initiation of Treatment    -   Established a baseline for a hibernating myocardium as        described, with perfusion determined by PET and cardiac function        by DSE immediately.    -   PRE-TREATMENT (under anesthesia):        -   Recorded baseline heart rate (HR)/blood pressure (BP)        -   Collected blood for:            -   serum chemistries, CBC, cardiac enzymes, such as CPK MB,                cardiac troponin I (“TNI”) or cardiac troponin T                (“TNT”), associated with damaged cardiac myocytes            -   spun plasma for rFGF-2 assay pre-treatment (freeze at                −70° C.)        -   EKG (3 leads, with rhythm strip)    -   DURING TREATMENT:        -   Recorded HR and BP data; treat hypotension with fluids        -   Recorded rhythm changes per monitor        -   Treated the four groups with FGF-2 (mid and high dose), a            negative control, and a positive control as described above.    -   POST-TREATMENT:        -   Recorded HRIBP until back to baseline        -   Collected second set of serum chemistries, CBC, cardiac            enzymes and spun plasma for rFGF-2 assay at latest possible            point post-treatment (2 hrs minimum) USE SAME TIME            post-treatment for blood collection in all animals. See            above for handling.        -   EKG (3 leads, with rhythm strip)            Phase 2: Follow-Up @ 3 Months Post-Treatment    -   Under anesthesia:        -   Recorded HR and BP        -   Collected blood for serum chemistries, CBC, cardiac enzymes            and spun plasma for rFGF-2 assay. See above for handling        -   Performed EKG (3 leads, with rhythm strip)        -   Determined perfusion by PET and myocardial function at            stress by DSE. Treatment group blinded to 2 readers            Phase 3: Histology and Final Report    -   Post-sacrifice: The minipigs were sacrificed 3 months after        treatment with FGF-2 or control.        -   Gross pathology of hearts: Recorded evidence of injection            site or other cardiac pathology (infarcts, scar, injection            site changes, pericardial changes)        -   Tissues: Septum, Anterior wall, LCx territory            -   Stained with hematoxylin and eosin (H&E) for                architecture            -   Stained with trichrome for fibrosis            -   Stained for alkaline phosphatase to identify endothelial                tissue                -   Performed a blinded assessment of overall vascular                    density in mid-myocardium cross-section                -   Searched for local pathology at injection sites                    (fibrosis, vascularity, myocyte loss, infarction,                    etc.)

The normalized perfusion ratio of the treated ischemic myocardium wasdetermined by PET three months after treatment with positive or negativecontrols (as described above) and after treatment IMc with the “mid”(0.6 μg/kg (13.5 μg)) or “high” 6.0 μg/kg (135 μg)) doses of rFGF-2 (SEQID NO: 2). This data is shown as the bar graph of FIG. 7, which alsocombines the normalized perfusion data from the “low” (0.06 μg/kg (1.35μg)) dose of FGF-2 as determined in Example 7. FIG. 7 shows that thegreatest % change in normalized perfusion (i.e., a 27.5% increase)surprisingly occurred for the “mid” dose, with the “low” and “high”doses showing lower changes of 17.5% and 17%, respectively. The data inFIG. 7 is the result of two separate experiments (light bars and darkbars) with the light colored placebo designated as “uld” (ultra-lowdose) being the placebo for the “low” dose, also shown as a lightcolored bar.

The % change in normalized perfusion for ischemic myocardium treatedwith the mid and high dose groups at 1 and 3 months after treatment iscompared to positive (IC) and negative (placebo) controls in the bargraph of FIG. 8. The “high” dose showed a higher increase in normalizedperfusion than was achieved for the “mid” dose at 1 monthpost-treatment. However, the % increase in normalized perfusionunexpectedly occurred for the “mid” dose of rFGF-2 IMc at 3 months posttreatment. This unexpectedly superior result is corroborated by theunexpectedly greater vascular density that was observed for the “mid”dose treated group than for the “high” dose treated group. (FIG. 9)Moreover, both showings of unexpectedly superior results for the middose treated group are consistent with the unexpectedly superiorupregulation of intracellular FGF-2 in the treated ischemic myocardiumthat is observed three months after treatment in the “mid” dose (about290 pg/ml) relative to that observed in the “high” dose group (about 170pg/ml) or in the positive IC control (about 175 pg/ml).

Thus, while all dosages of FGF-2 that are administered IMc in accordancewith the method of the present invention increase perfusion and cardiacfunction, there appears to be an unexpectedly superior (mid) dosage ofFGF-2 that occurs from about 0.3 μg/kg (or 6.75 μg or 6,750 ng) to about3.0 μg/kg (or 67.5 μg or 67,500 ng).

1. A method for inducing angiogenesis in a heart of a patient,comprising injecting an effective amount of an angiogenic agent directlyinto the myocardium of said patient in one or more areas in need ofangiogenesis, said effective amount of angiogenic agent being from about5 ng to less than 135,000 ng of said angiogenic agent.
 2. The method ofclaim 1, wherein said angiogenic agent is platelet derived growth factor(PDGF), vascular endothelial growth factor-A (VEGF-A), VEGF-D, afibroblast growth factor (FGF), or an angiogenically active fragment ormutein thereof.
 3. The method of claim 2, wherein said effective amountof angiogenic agent is from 5 ng to 67,500 ng of said angiogenic agent.4. The method of claim 2, wherein said angiogenic agent is an FGF or anangiogenically active fragment or mutein thereof, and said FGF is FGF-2.5. The method of claim 4, wherein said FGF-2 has the amino acid sequenceof SEQ ID NO:2,5, or
 6. 6. The method of claim 1, wherein said patienthas symptoms of coronary artery disease (CAD) or a myocardial infarction(MI).
 7. A method for treating a human patient for coronary arterydisease, comprising injecting an effective amount of an angiogenic agentdirectly into the myocardium in one or more areas in need of treatmentfor said disease, said effective amount of angiogenic agent being fromabout 5 ng to less than 135,000 ng of said angiogenic agent.
 8. Themethod of claim 7, wherein said angiogenic agent is platelet derivedgrowth factor (PDGF), vascular endothelial growth factor-A (VEGF-A),VEGF-D, a fibroblast growth factor (FGF), or an angiogenically activefragment or mutein thereof.
 9. The method of claim 8, wherein saideffective amount of angiogenic agent is from 5 ng to 67,500 ng of saidangiogenic agent.
 10. The method of claim 8, wherein said angiogenicagent is an FGF or an angiogenically active fragment or mutein thereof,and said FGF is FGF-2.
 11. The method of claim 10, wherein said FGF-2has the amino acid sequence of SEQ ID NO: 2,5, or
 6. 12. A method forincreasing vascular perfusion or vascular density in the myocardiumcomprising injecting an area of the myocardium in need of an increase inperfusion or vascular density with an effective amount of an angiogenicagent, said effective amount being within the range of about 5 ng toless than 135,000 ng of said angiogenic agent.
 13. The method of claim12, wherein said angiogenic agent is platelet derived growth factor(PDGF), vascular endothelial growth factor-A (VEGF-A), VEGF-D, afibroblast growth factor (FGF), or an angiogenically active fragment ormutein thereof.
 14. The method of claim 13, wherein said effectiveamount of angiogenic agent is from 5 ng to 67,500 ng of said angiogenicagent.
 15. The method of claim 13, wherein said angiogenic agent is anFGF or an angiogenically active fragment or mutein thereof, and said FGFis FGF-2.
 16. A method for stimulating the production of FGF-2 and VEGFin human myocardial cells for up to three months, comprising injectingan effective amount of an angiogenic agent directly into the myocardiumof said patient in one or more areas in need of angiogenesis, saideffective amount of angiogenic agent being from about 5 ng to less than135,000 ng of said angiogenic agent.
 17. The method of claim 16, whereinsaid effective amount of angiogenic agent is 6.75 μg to 67.5 μg of saidangiogenic agent.
 18. A pharmaceutical composition comprising aneffective amount of an angiogenic agent in a pharmaceutically acceptablecarrier, said effective amount of angiogenic agent being in the rangefrom about 5 ng to less than about 135,000 ng of said angiogenic agent.19. The pharmaceutical composition of claim 18, wherein said angiogenicagent is platelet derived growth factor (PDGF), vascular endothelialgrowth factor-A (VEGF-A), VEGF-D, a fibroblast growth factor (FGF), oran angiogenically active fragment or mutein thereof.
 20. Thepharmaceutical composition of claim 19, wherein said effective amount ofangiogenic agent is in the range from 5 ng to 67,500 ng of saidangiogenic agent.